Pharmaceutical compositions and methods useful for modulating angiogenesis, inhibiting metastasis and tumor fibrosis, and assessing the malignancy of colon cancer tumors

ABSTRACT

Methods and compositions suitable for modulating angiogenesis in a mammalian tissue are provided. Further provided are methods suitable for inhibiting metastasis and fibrosis in a mammalian tissue and for assessing the malignancy of colon cancer tumors.

RELATED PATENT APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/647,017, filed 11 Jul. 2017, pending, which application is acontinuation of U.S. patent application Ser. No. 14/311,110, filed 20Jun. 2014, now abandoned, which application is a continuation of U.S.patent application Ser. No. 13/412,544, filed 5 Mar. 2012, nowabandoned, which application is a continuation of U.S. patentapplication Ser. No. 10/536,440, filed 14 Nov. 2005, now issued as U.S.Pat. No. 8,163,494, which is a national phase application ofPCT/IL03/01008 having an international filing date of 27 Nov. 2003,which claims benefit of U.S. patent application Ser. No. 10/305,348,filed 27 Nov. 2002, now abandoned. The entire contents of theseapplications are incorporated herein by this reference.

REFERENCE TO SEQUENCE LISTING SUBMITTED VIA EFS-WEB

The Sequence Listing associated with this application is provided intext format in lieu of a paper copy, and is hereby incorporated byreference into the specification. The name of the text file containingthe Sequence Listing is GILE-051_09US_ST25.txt. The text file is 45 KB,was created on Jan. 26, 2018, and is being submitted electronically viaEFS-Web.

FIELD AND BACKGROUND OF THE INVENTION

The present invention relates to pharmaceutical compositions and methodsuseful for modulating angiogenesis and for inhibiting metastasis andfibrosis in a mammalian tissue. The present invention further relates toa method of assessing the malignancy of colon tumors and predicting theprognosis of colon cancer.

Angiogenesis

In an adult, formation of new blood vessels in normal or diseasedtissues is regulated by two processes, recapitulated vasculogenesis (thetransformation of pre-existing arterioles into small muscular arteries)and angiogenesis, the sprouting of existing blood vessels (which occursboth in the embryo and in the adult).

The process of angiogenesis is regulated by biomechanical andbiochemical stimuli. Angiogenic factors such as vascular endothelialgrowth factor (VEGF) and basic fibroblast growth factor (bFGF) arereleased by vascular cells, macrophages, and cells surrounding bloodvessels. These angiogenic factors activate specific proteases that areinvolved in degradation of the basement membrane. As a result of thisdegradation, vascular cells migrate and proliferate thus leading to newblood vessel formation. Peri-endothelial cells, such as pericytes in thecapillaries, smooth muscle cells in larger vessels and cardiac myocytesin the heart are recruited to provide maintenance and modulatoryfunctions to the forming vessel.

The establishment and remodeling of blood vessels is controlled byparacrine signals, many of which are mediated by protein ligands whichmodulate the activity of transmembrane tyrosine kinase receptors. Amongthese molecules are vascular endothelial growth factor (VEGF) and itsreceptor families (VEGFR-1, VEGFR-2, neuropilin-1 and neuropilin-2),Angiopoietins 1-4 (Ang-1, Ang-2 etc.) and their respective receptors(Tie-1 and Tie-2), basic fibroblast growth factor (bFGF), plateletderived growth factor (PDGF), and transforming growth factor β (TGF-β).

The growth of solid tumors is limited by the availability of nutrientsand oxygen. When cells within solid tumors start to produce angiogenicfactors or when the levels of angiogenesis inhibitors decline, thebalance between anti-angiogenic and angiogenic influences is perturbed,initiating the growth of new blood vessels from the existing vascularbed into the tumor. This event in tumor progression is known as theangiogenic switch (Folkman, 1990; Hanahan and Folkman 1996). It had beendemonstrated that inhibitors of tumor angiogenesis are able tocompletely inhibit tumor growth in mice (Boehm et al., 1997; Bergers etal., 1999) and also inhibit tumor metastasis, a process that relies uponclose contact between the vasculature and tumor cells (Zetter, 1998). Ithas also been demonstrated that angiogenesis plays an important role inthe progression of breast cancer (Weidner, N. 1998; Degani et al., 1997;Guidi et al., 1997; Balsari et al., 1999).

Such findings have prompted the use of known anti-angiogenic factors inbreast cancer therapy (Klauber et al., 1997; Harris et al., 1996;Weinstatsaslow et al., 1994) and a search for novel angiogenesisinhibitors.

During the past decade several novel inhibitors of angiogenesis havebeen isolated including inhibitors of VEGF signaling (Neufeld et al.,1999) and inhibitors of processes which lead to the maturation andstabilization of new blood vessels. Anti-integrin antibodies have beenused as inhibitors of blood vessel maturation (Brooks et al., 1994;Brooks et al., 1998).

Although several anti-angiogenic drugs are now available commercially,the anti-angiogenic mechanisms of most of these drugs (e.g., angiostatinand endostatin) remain unclear (O'Reilly et al., 1997; Oreilly et al.,1996).

Since angiogenesis can be initiated by many (possibly compensatory)angiogenic factors it stands to reason that anti-angiogenic factorswhich target later processes in the angiogenic response such as vesselmaturation or a combination of anti-angiogenic factors would be mosteffective in arresting vessel formation.

Platelet factor-4 (PF4) is an anti-angiogenic protein normallysequestered in platelets (Tanaka et al., 1997; Maione et al., 1990;Neufeld et al., 2000). PF4 inhibits angiogenesis using poorly definedmechanisms (Gengrinovitch et al., 1995; Brown, and Parish, 1994; Gupta,and Singh, 1994; Watson et al., 1994). It was previously speculated thatPF4 binds to cell surface heparan-sulfate proteoglycans and in thismanner inhibits the activity of angiogenic growth factors such as basicfibroblast growth factor (Watson et al., 1994).

Tumor Metastasis and Staging

The transition from a localized tumor to an invasive and metastatictumor represents a landmark in the development of malignant disease,since it is usually associated with a markedly worse prognosis. Theunderstanding of the processes that govern this transition is thereforeof prime importance.

Breast Cancer

In breast cancer, the transition from a localized to aninvasive/metastatic tumor is associated in many cases with the formationof fibrotic foci and desmoplasia, which is the presence of unusuallydense collagenous stroma, within the primary tumor (Colpaert et al.,2001; Hasebe et al., 2000). A similar correlation may exist in othertypes of cancers such as colon and pancreatic cancers (Nishimura et al.,1998; Ellenrieder et al., 2000). These observations represent apparentparadoxes at first glance, since invasiveness has long been associatedwith the destruction of extracellular matrix by extracellular matrixdegrading enzymes like metalo-proteases (Stamenkovic, 2000; Duffy etal., 2000) and heparanase (Vlodaysky and Friedmann, 2001). However, itis possible that deposition of excess extracellular matrix may stimulatein turn expression of matrix degrading enzymes that will contributeunder certain circumstances to tumor invasion. In fact, there is someevidence that an increase in extracellular matrix deposition can indeedinfluence the production of extracellular matrix degrading enzymes(Schuppan et al., 2001; Swada et al., 2001).

Several prior art studies have attempted to develop agents to treatbreast cancer metastases (Sauer et al., 2002) including a study by Kimet al., (2000) that described apicidin [cyclo(N-O-methyl-L-tryptophanyl-L-isoleucinyl-D-pipecolinyl-L-2-amino-8-oxodecanoyl)],a fungal metabolite that was identified as an antiprotozoal agent knownto inhibit parasite histone deacetylase (HDAC), that can inhibit theH-ras-induced invasive phenotype of MCF10A human breast epithelialcells. Another agent is the polymeric form of fibronectin that was shownto reduce tumor growth and to posses antimetastatic activity whenadministered systemically to tumor-bearing mice (Yi and Ruoslahti,2001).

Colon Cancer

Cancer of the gastrointestinal (GI) tract, especially colon cancer, is ahighly treatable and often a curable disease when localized to thebowel. Surgery is the primary treatment and results in cure inapproximately 50% of patients. Recurrence following surgery is a majorproblem and often is the ultimate cause of death. Nearly all cases ofcolorectal cancer arise from adenomatous polyps, some of which matureinto large polyps, undergo abnormal growth and development, andultimately progress into cancer. This progression would appear to takeat least 10 years in most patients, rendering it a readily treatableform of cancer if diagnosed early, when the cancer is localized.

The standard procedures currently used for establishing a definitivediagnosis for a GI tract cancer include barium studies, endoscopy,biopsy and computed tomography [M. F. Brennan, et al. In: Cancer:Principles and Practice of Oncology, Fourth Edition, pp. 849-882,Philadelphia, Pa.: J. B. Lippincott Co. (1993)].

The prognosis of colon cancer is clearly related to the degree ofpenetration of the tumor through the bowel wall and the presence orabsence of nodal involvement. These two characteristics form the basisfor all staging systems developed for this disease. Staging is usuallyperformed by a pathologist on tissue sections obtained via biopsy and/orsurgery and it aims to determine the anatomic extent of the disease.Accurate staging is critical for predicting patient outcome andproviding criteria for designing optimal therapy. Inaccurate staging canresult in poor therapeutic decisions and is a major clinical problem incolon cancer.

Thus, to increase the accuracy of therapy and the survival rate of coloncancer patients there is a need to develop sensitive and accuratemethods of staging of colon cancer.

SUMMARY OF THE INVENTION

While reducing the present invention to practice, the present inventorshave uncovered a novel PF4 binding protein, a lysyl oxidase protein,LOR-1, which participates in modulating angiogenesis and breast cancermetastases and as such can be used as a target for inhibiting metastasisand reducing tumor invasiveness. Moreover, the present inventors haveuncovered that the expression of LOR-1 is correlated with colon cancerprogression and as such can be used for accurate staging of coloncancer.

According to one aspect of the present invention there is provided amethod of modulating angiogenesis in a mammalian tissue, the methodcomprising administering into the mammalian tissue a molecule capable ofmodifying a tissue level and/or activity of at least one type of lysyloxidase to thereby modulate angiogenesis in the mammalian tissue.

According to another aspect of the present invention there is provided amethod of modulating angiogenesis in a mammalian tissue, the methodcomprising administering into the mammalian tissue a nucleic acidconstruct being capable of expressing a polypeptide having lysyl oxidaseactivity to thereby modulate angiogenesis within the mammalian tissue.

According to yet another aspect of the present invention there isprovided a pharmaceutical composition useful for modulating angiogenesisin mammalian tissue comprising, as an active ingredient, a moleculecapable of modifying a level and/or activity of at least one type oflysyl oxidase of the mammalian tissue and a pharmaceutically effectivecarrier.

According to another aspect of the present invention there is provided amethod of modulating angiogenesis in a mammalian tissue, the methodcomprising administering into the mammalian tissue a molecule capable ofmodifying a tissue level and/or activity of a polypeptide at least 75%homologous to the polypeptide set forth in SEQ ID:2 or 9 to therebymodulate angiogenesis within the mammalian tissue. According to furtherfeatures in preferred embodiments of the invention described below, themolecule is an antibody or an antibody fragment capable of binding with,and at least partially inhibiting the activity of, the at least onepolypeptide.

According to still further features in the described preferredembodiments the antibody or the antibody fragment is directed against atleast a portion of the polypeptide set forth in SEQ ID NO:2, 3, 6, 8 or9.

According to still further features in the described preferredembodiments the molecule is a polynucleotide capable of down regulatingexpression of the at least one type of lysyl oxidase.

According to still further features in the described preferredembodiments the polynucleotide is at least partially complementary withthe polynucleotide set forth in SEQ ID NO:1, 4, 5 or 7.

According to still further features in the described preferredembodiments the molecule is a polypeptide having lysyl oxidase activity.

According to still further features in the described preferredembodiments the polypeptide is as set forth in SEQ ID NO:2, 3, 6, 8 or9.

According to another aspect of the present invention there is provided amethod of modulating angiogenesis in a mammalian tissue, the methodcomprising administering into the mammalian tissue a nucleic acidconstruct being capable of expressing a polypeptide having lysyl oxidaseactivity to thereby modulate angiogenesis within the mammalian tissue.

According to still further features in the described preferredembodiments the polypeptide is at least 75% homologous to thepolypeptide set forth in SEQ ID NO:2, 3, 6, 8 or 9.

According to still another aspect of the present invention there isprovided method of identifying molecules capable of modulatingangiogenesis, the method comprising: (a) isolating molecules whichexhibit specific reactivity with at least one type of lysyl oxidase; and(b) testing the molecules within mammalian tissue so as to determine theangiogenesis modulation activity thereof.

According to still further features in the described preferredembodiments step (a) is effected by binding assays and/or lysyl oxidaseactivity assays.

According to an additional aspect of the present invention there isprovided method of determining the malignancy of cancerous tissue, themethod comprising (a) determining a lysyl oxidase expression leveland/or activity of the cancerous tissue; and (b) comparing the lysyloxidase expression level and/or activity with that determined forcontrol tissue to thereby determine the malignancy of the canceroustissue.

According to another aspect of the present invention there is providedmethod of inhibiting metastasis and fibrosis in a mammalian tissue, themethod comprising administering into the mammalian tissue a moleculecapable of downregulating a tissue level and/or activity of at least onetype of lysyl oxidase to thereby inhibit metastasis in the mammaliantissue

According to still another aspect of the present invention there isprovided a pharmaceutical composition useful for inhibiting metastasisand fibrosis in mammalian tissue comprising, as an active ingredient, amolecule capable of downregulating a level and/or activity of at leastone type of lysyl oxidase of the mammalian tissue and a pharmaceuticallyeffective carrier.

According to further features in preferred embodiments of the inventiondescribed below, the molecule is an antibody or an antibody fragmentcapable of binding with, and at least partially inhibiting the activityof, the at least one polypeptide.

According to still further features in the described preferredembodiments the antibody or the antibody fragment is directed against atleast a portion of the polypeptide set forth in SEQ ID NO:2, 3, 6, 8 or9.

According to still further features in the described preferredembodiments the molecule is a polynucleotide capable of downregulatingexpression of the at least one type of lysyl oxidase.

According to still further features in the described preferredembodiments the polynucleotide is at least partially complementary withthe polynucleotide set forth in SEQ ID NO:1, 4, 5 or 7.

According to still another aspect of the present invention there isprovided method of identifying molecules capable of inhibitingmetastasis and fibrosis, the method comprising: (a) screening andidentifying molecules which exhibit specific reactivity with at leastone type of lysyl oxidase; and (b) testing the metastasis and fibrosisinhibitory potential of the said molecules.

According to still further features in the described preferredembodiments step (a) is effected by binding assays and/or lysyl oxidaseactivity assays.

According to yet another aspect of the present invention there isprovided a method for inhibiting metastasis and fibrosis in a mammaliantissue, the method comprising administering to the mammalian tissue amolecule capable of downregulating a tissue level and/or activity of apolypeptide at least 75% homologous to the polypeptide set forth in IDNO: 2 or 9, to thereby inhibit metastasis and fibrosis in a mammaliantissue.

According to yet another aspect of the present invention there isprovided a method of assessing a malignancy of a colon tumor comprisingdetermining a tissue level and/or an activity level of a polypeptide atleast 75% homologous to the polypeptide set forth in SEQ ID NO:2 or 9 ina tissue of the colon tumor, thereby assessing the malignancy of thecolon tumor.

According to yet another aspect of the present invention there isprovided a method of predicting a prognosis of an individual diagnosedwith colon cancer comprising: (a) obtaining a colon tumor tissue fromthe individual, and; (b) determining a tissue level and/or an activitylevel of a polypeptide at least 75% homologous to the polypeptide setforth in SEQ ID NO:2 or 9 in the colon tumor tissue to thereby assessthe malignancy of the colon tumor tissue and predict the prognosis ofthe individual diagnosed with colon cancer.

According to still further features in the described preferredembodiments the tissue of the colon tumor is obtained using a colonbiopsy and/or a colon surgery.

According to still further features in the described preferredembodiments determining the tissue level of the polypeptide is effectedby an immunological detection method and/or an RNA detection method.

According to still further features in the described preferredembodiments determining the activity level of the polypeptide iseffected by an enzymatic activity detection method.

According to still further features in the described preferredembodiments the immunological detection method is selected from thegroup consisting of a radio-immunoassay (RIA), an enzyme linkedimmunosorbent assay (ELISA), a Western blot analysis, and animmunohistochemical analysis.

According to still further features in the described preferredembodiments the RNA detection method is selected from the groupconsisting of a Northern blot analysis, an RNA in situ hybridizationstain, an RT-PCR analysis, and an in situ RT-PCR stain.

According to still further features in the described preferredembodiments the enzymatic activity detection method is selected from thegroup consisting of a cytochemical stain, an in vitro activity assay,and an activity gel.

According to still further features in the described preferredembodiments the malignancy of the colon tumor is assessed by comparingthe tissue level and/or the activity level of the polypeptide in thetissue of the colon tumor with a tissue level and/or activity level ofthe polypeptide in a normal colon tissue.

The present invention successfully addresses the shortcomings of thepresently known configurations by providing pharmaceutical compositionsand methods that can be used to treat metastatic cancer, formation oftumor fibrosis and other disorders characterized by excessive orinsufficient blood vessel formation as well as by providing a method ofassessing the malignancy of colon cancer.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. In case of conflict, the patentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is herein described, by way of example only, withreference to the accompanying drawings. With specific reference now tothe drawings in detail, it is stressed that the particulars shown are byway of example and for purposes of illustrative discussion of thepreferred embodiments of the present invention only, and are presentedin the cause of providing what is believed to be the most useful andreadily understood description of the principles and conceptual aspectsof the invention. In this regard, no attempt is made to show structuraldetails of the invention in more detail than is necessary for afundamental understanding of the invention, the description taken withthe drawings making apparent to those skilled in the art how the severalforms of the invention may be embodied in practice.

In the drawings:

FIG. 1 illustrates SDS-PAGE analysis of extracts of Porcine aorticendothelial cells (PAE Cells) which were transfected with vector along(lane 1) or with vector containing the LOR-1 cDNA (lane 3) andmetabolically labeled with ³⁵5-methionine. Extracts from the vectortransfected cells (lane 2), from vector containing LOR-1 cDNAtransfected cells (lane 4) or from ³⁵S-methionine labeled humanumbilical vein endothelial cells (HUVEC) (lane 5) were purified on a PF4affinity column. A Band corresponding in size to the original bandobserved in the HUVEC is evident (compare lanes 4 and 5); this band isabsent in extracts of vector transfected cells.

FIG. 2 illustrates differential expression of LOR-1 in breast cancerderived cells of different metastatic potential. The metastaticpotential of the cells increases from left to right, and is correlatedto increased LOR-1 mRNA expression. The results correspond to northernblot analysis of LOR-1 mRNA expression. Data regarding the relativemetastatic potential of the cell lines was derived from the literature.

FIG. 3 illustrates expression of recombinant LOR-1 in MCF-7 breastcancer cells (lane 1). Vector transfected MCF-7 cells (lane 2) and twoclones of MCF-7 expressing recombinant LOR-1 (lane 3, clone 12, lane 4,clone 22) were grown for two days in serum free medium. The medium froman equal number of cells was collected, concentrated 30 fold usingCentricon™, and 10 μl aliquots were electrophoresed using an SDS/PAGEgel. Proteins were blotted onto nitrocellulose, and LOR-1 protein wasidentified using an antibody directed against the C-terminal of LOR-1. Asecondary alkaline-phosphatase coupled antibody and NBT-BICP stainingwere used to detect the primary bound antibody.

FIG. 4 illustrates tumor size as correlated to LOR-1 expression.Parental MCF-7 cells (par), MCF-7 cells transfected with pCDNA3 vectoralone (vec) and two recombinant LOR-1 expressing MCF-7 cells (clones 12and 24) were deposited under the skin of immune deficient mice(10⁷/injection site) along with an estrogen slow release pellet. Sixanimals were used for each cell type implanted. The area of the tumorswas measured every few days. Bars represent standard deviation from themean.

FIGS. 5a-b illustrate anti-factor-8 immunostaining of tumors generatedby MCF-7 cells transfected with the expression vector alone (FIG. 5a )or with an expression vector containing the LOR-1 cDNA (FIG. 5b ).Counter staining was performed with Hematoxylin-eosin (blue). Invasionof blood vessels into the tumor mass is more abundant in LOR-1expressing tumors (FIG. 5b ) as compared to tumors generated by controlcells which do not express LOR-1 (FIG. 5a ).

FIGS. 6a-d illustrate liver sections of Wilson's disease patients (FIGS.6c-d ) and normal patients (FIGS. 6a-b ) probed with a LOR-1 sense probe(FIGS. 6a, 6c ) and antisense probe (FIGS. 6b, 6d ).

FIG. 7 illustrates results of a whole mount in-situ hybridization usinga LOR-1 cDNA probe and a 4 day old chick embryo. Strong expression ofLOR-1 mRNA is observed in amniotic blood vessels (arrow).

FIG. 8 illustrates sequence alignment of several lysyl oxidasesincluding LOR-1.

FIGS. 9a -b. illustrate the expression of LOR-1 and LOR-2 in humanbreast cancer derived cells: Total RNA was prepared from confluent MCF-7cells (MCF-7), MDA-MB-231 cells (MDA-231) and MDA-MB-435 cells (MDA-435)and was subjected to Northern blot analysis using a LOR-1 (FIG. 9a ) andLOR-2 (FIG. 9b ) specific cDNA probes. LOR-1 specific hybridizationsignal is seen in tumors derived from both MDA-231 and MDA-435 cells butnot in tumors derived from MCF-7 cells. LOR-2 specific hybridizationsignal is seen only in tumors derived from MDA-435 cells. LOR-1 andLOR-2 non-specific hybridization signals are seen in all tumors' RNA ina band that corresponds to the 28S rRNA.

FIGS. 9c-f illustrate the expression pattern of LOR-1 in normal humanbreast (FIG. 9c ), in in-situ non-invasive breast carcinoma (FIG. 9d ),in grade-1 invasive ductal carcinoma (FIG. 9e ) and in grade-3 invasiveductal carcinoma (FIG. 9f ). A polyclonal affinity purified rabbitantibody directed against the C-terminal of LOR-1 was used to detect theexpression of LOR-1. High level expression of LOR-1 protein is seen inthe epithelium of the normal duct (FIG. 9c , open arrow); magnification×100. In in-situ non-invasive breast carcinoma (FIG. 9d ) the cancercells have filled the duct but are still confined to it. Many of thecells located at periphery of the tumor have lost their ability toexpress LOR-1, while at the center, the cells still express high levelsof LOR-1 (FIG. 9d , empty arrow); magnification ×200. In grade-1invasive breast carcinoma the tumorigenic cells have formed pseudo-ducts(FIG. 9e , black arrows) but they do not express LOR-1 anymore. However,cells found in a nearby carcinoma in-situ express LOR-1 (FIG. 9e , emptyarrow); magnification ×200. In grade-3 invasive breast carcinoma thetumorigenic cells express large amounts of LOR-1 and the morphology iscompletely disorderly (FIG. 9f ); magnification ×200.

FIG. 10a illustrates the expression of recombinant LOR-1 in MCF-7 cellstransfected with the expression vector alone (vec) or with an expressionvector containing the LOR-1 cDNA clone 12 or 24 (#12 or #24,respectively). LOR-1 proteins were detected by Western blot with anantibody directed against the C-terminal of human LOR-1.

FIG. 10b illustrates post-translational processes of LOR-1.MCF-7/Tet-LOR1 cells grown in the presence of tetracycline weretrypsinized and seeded into a 24-well dish (5×10⁴ cells/well) in a serumfree medium in the absence of tetracycline. Cell aliquots collected atthe noted times following tetracycline removal and were analyzed for thepresence of LOR-1 by Western blot as described in FIG. 10 a.

FIG. 10c illustrates the growth rate of tumors derived from control orLOR-1 expressing MCF-7 cells. MCF-7 cells transfected with theexpression vector alone (vec) or with an expression vector containingthe LOR-1 cDNA clone 12 or 24 were injected into the mammary fat pads offemale athymic nude mice. Each cell type was implanted in 8 animals.Tumor area was measured at the indicated times. Error bars represent thestandard error of the mean. The experiment was terminated and the micesacrificed when the tumor reached a diameter of about 1 cm.

FIG. 10d illustrates the relative size of tumor in mice 25 daysfollowing injection of MCF-7 cells. Shown are mice harboring tumors thatdeveloped from cells transfected with the expression vector alone (left)or with the expression vector containing LOR-1 cDNA clone 24 (center) or12 (right).

FIGS. 11a-h illustrate fibrotic foci and collagen deposits in tumorsderived from MCF-7 cells expressing recombinant LOR-1. Hematoxylin-eosinstaining demonstrate a few necrotic foci in a tumor derived from MCF-7cells transfected with expression vector alone (FIG. 11a , arrow,magnification ×20) and numerous necrotic foci in a tumor derived fromMCF-7 cells transfected with expression vector containing LOR-1 cDNAclone 12 (FIG. 11b , arrows, magnification ×20). FIG. 11c illustrateshuman keratin-7 immunostaining of tumors generated by MCF-7 cellstransfected with expression vector containing clone 12. Counter stainingwas performed with Hematoxylin-eosin (blue). Arrow points to nuclei ofhost cells concentrated in the fibrotic foci. No necrosis can be seen;magnification ×40. FIGS. 11d-f illustrate collagen deposits in tumorcells as viewed by Masson's Trichrome stain. A few collagen deposits areseen in tumors generated from MCF-7 cells transfected with theexpression vector alone (FIG. 11d , arrows, magnification ×200). Thickcollagen bundles are seen between tumor cells generated from MCF-7 cellstransfected with expression vector containing clone 12 (FIG. 11 e,magnification ×200). The fibrotic area is full with collagen fibers andinterspaced with host derived cells in tumors generated from MCF-7 cellstransfected with expression vector containing clone 24 (FIG. 11 f,magnification ×200). FIGS. 11g-h illustrate blood vessels stained withMasson's Trichrome in tumors generated from MCF-7 cells transfected withthe expression vector alone (FIG. 11g ) or expression vector containingclone 12 (FIG. 11h ); magnification ×400.

FIGS. 12a-d illustrate the deposition of collagen type-3 by reticulumstain in tumors generated from MCF-7 and C6 glioma cells transfectedwith expression vector alone (FIGS. 12a, c ) or vector expressing LOR-1cDNA clone 12 (FIGS. 12b, d ); magnification ×200.

FIGS. 13a-h illustrate the invasiveness of tumors derived from MCF-7cells expressing recombinant LOR-1. Shown are histological sections oftumors generated from MCF-7 cells transfected with an expression vectoralone (FIGS. 13a, b ) or a vector expressing LOR-1 (FIGS. 13c-h ).Sections were labeled with either a monoclonal antibody specific tohuman keratin-7 (FIGS. 13a-d and f -h, blue purple stain) or with anantibody directed against LOR-1 (FIG. 13e , red stain). Counterstain waswith Hematoxylin (light blue) in all sections. Black arrows designatecytokeratin-7 positive tumor cells invading the tumor pseudo-capsule(FIG. 13c ), infiltrating between adjacent muscle bundles (FIG. 13d ),or invading the vasculature (FIGS. 13f, g ) and peri-neural space ofnerves (FIG. 13h ). White arrows designate LOR-1 positive tumor cellsinfiltrating muscles located next to the tumor (FIG. 13g ). Bloodvessels v; nerves n; muscle fibers m; magnification ×100 for FIGS. 13a-c, f, h and ×200 for FIGS. 13 d, e, g.

FIGS. 14a-d illustrate the expression pattern of LOR-1 in normal humancolon (FIG. 14a ), in a colon tumor including hyperplasia (FIG. 14b ,diamond arrows) and adenoma (FIG. 14b , circled arrows), in a low-gradecolon adenocarcinoma (FIG. 14c , arrows, brown stain) and in ahigh-grade colon adenocarcinoma (FIG. 14d , brown stain). A polyclonalaffinity purified rabbit antibody directed against the C-terminal ofLOR-1 was used to detect the expression of LOR-1 by immunohistochemistryin tissue sections obtained from various colon cancer tumors. Note thefaint LOR-1 staining in a few cells of the normal colon (FIG. 14a ,arrows, magnification ×10), the moderate LOR-1 staining in hyperplasiatumor (FIG. 14b , diamond circles) and the high LOR-1 staining in cellsof the colon adenoma (FIG. 14b , circle arrows, magnification ×4). Alsonote the significant LOR-1 staining in low-grade adenocarcinoma (FIG.14c , arrows, magnification ×10) and in high-grade carcinoma (FIG. 14d ,magnification ×20).

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is of pharmaceutical compositions and methods thatcan be used to modulate angiogenesis and to inhibit tumor invasivenessand tumor fibrosis. Specifically, the present invention can be used tosuppress tumor growth and metastasis as well as to treat disorders suchas, for example, arthritis, diabetic retinopathy, psoriasis andvasculitis. Moreover, the present invention is of a method ofdetermining the malignancy of colon cancer tumors which can be used forcolon cancer staging. Specifically, the present invention can be used topredict the prognosis of colon cancer patients based on the tissue leveland/or activity level of LOR-1 in the colon tumors.

The principles and operation of the present invention may be betterunderstood with reference to the drawings and accompanying descriptions.

Before explaining at least one embodiment of the invention in detail, itis to be understood that the invention is not limited in its applicationto the details of construction and the arrangement of the components setforth in the following description or illustrated in the drawingsdescribed in the Examples section. The invention is capable of otherembodiments or of being practiced or carried out in various ways. Also,it is to be understood that the phraseology and terminology employedherein is for the purpose of description and should not be regarded aslimiting.

As described in the Examples section which follows, the presentinventors have uncovered a novel protein constituent of the angiogenicprocess.

This protein, which is termed LOR-1 herein (SEQ ID NO:2) belongs to thelysyl oxidase family of enzymes which catalyze the formation of covalentcrosslinks between lysine residues on adjacent collagen or elastinfibrils. The lysyl oxidase family includes four genes (Saito et al.,1997; Kim et al., 1995; Reiser et al., 1992; Jang et al., 1999), theprotein sequences of which are presented in SEQ ID NOs:3, 6, 8 and 9. Ahomology comparison between several lysyl oxidase family members ispresented in FIG. 8 which is further described in the Examples sectionwhich follows.

Each member of the lysyl oxidase family of enzymes includes a highlyconserved lysyl oxidase domain, the activity of which is highlydependent on the presence of copper.

It should be noted that prior art studies have shown that removal ofcopper from tumor tissues leads to inhibition of angiogenesis(Rabinovitz, 1999; Yoshida et al., 1995). This further substantiates therole of the lysyl oxidase family of enzymes in angiogenesis sincepresumably, removal of copper leads to inhibition of lysyl oxidases.

Further support to the angiogenic activity of lysyl oxidases is providedby the PF4-LOR-1 binding assays presented herein. As mentionedhereinabove, PF4 is an inhibitor of angiogenesis. As such, theanti-angiogenic activity exhibited by PF4 may be effected through LOR-1inhibition, which, as demonstrated in the Examples section whichfollows, is highly expressed in the endothelial cells lining bloodvessels.

Thus according to one aspect of the present invention there is provideda method of modulating angiogenesis

The method is effected by administering into the mammalian tissue amolecule capable of modifying a tissue level and/or activity of at leastone type of lysyl oxidase to thereby modulate angiogenesis in themammalian tissue.

As used herein, the phrase “tissue level” refers to the level of lysyloxidase protein present in active form in the tissue at a given timepoint. Protein levels are determined by factors such as, transcriptionand/or translation rates, RNA or protein turnover and/or proteinlocalization within the cell. As such any molecule which effects any ofthese factors can modify the tissue level of the lysyl oxidase.

As used herein the term “activity” refers to an enzymatic activity ofthe lysyl oxidase. A molecule which can modify the enzymatic activitymay directly or indirectly alter substrate specificity of the enzyme oractivity of the catalytic site thereof.

There are numerous examples of molecules which can specifically modifythe tissue level and/or activity of a lysyl oxidase. Such molecules canbe categorized into lysyl oxidase “downregulators” or “upregulators”.

Downregulators

One example of an agent capable of downregulating a lysyl oxidaseprotein is an antibody or antibody fragment capable of specificallybinding lysyl oxidase or at least part of the lysyl oxidase protein(e.g., region spanning the catalytic site) and inhibiting its activitywhen introduced into the mammalian tissue. As such, an antibody or anantibody fragment directed at a lysyl oxidase can be used to suppress orarrest the formation of blood vessels, and to inhibit tumor fibrosis andmetastasis.

Numerous examples of antibody inhibitors are known in the art, includinginhibitors of angiogenesis which target angiogenic factors (Brooks etal., 1994; Brooks et al., 1998).

Preferably, the antibody specifically binds to at least one epitope of alysyl oxidase. As used herein, the term “epitope” refers to anyantigenic determinant on an antigen to which the paratope of an antibodybinds.

Epitopic determinants usually consist of chemically active surfacegroupings of molecules such as amino acids or carbohydrate side chainsand usually have specific three-dimensional structural characteristics,as well as specific charge characteristics.

The term “antibody” as used in this invention includes intact moleculesas well as functional fragments thereof, such as Fab, F(ab′)2, and Fvthat are capable of binding to macrophages. These functional antibodyfragments are defined as follows: (1) Fab, the fragment which contains amonovalent antigen-binding fragment of an antibody molecule, can beproduced by digestion of whole antibody with the enzyme papain to yieldan intact light chain and a portion of one heavy chain; (2) Fab′, thefragment of an antibody molecule that can be obtained by treating wholeantibody with pepsin, followed by reduction, to yield an intact lightchain and a portion of the heavy chain; two Fab′ fragments are obtainedper antibody molecule; (3) (Fab′)2, the fragment of the antibody thatcan be obtained by treating whole antibody with the enzyme pepsinwithout subsequent reduction; F(ab′)2 is a dimer of two Fab′ fragmentsheld together by two disulfide bonds; (4) Fv, defined as a geneticallyengineered fragment containing the variable region of the light chainand the variable region of the heavy chain expressed as two chains; and(5) Single chain antibody (“SCA”), a genetically engineered moleculecontaining the variable region of the light chain and the variableregion of the heavy chain, linked by a suitable polypeptide linker as agenetically fused single chain molecule.

Methods of producing polyclonal and monoclonal antibodies as well asfragments thereof are well known in the art (See for example, Harlow andLane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory,New York, 1988, incorporated herein by reference).

Antibody fragments according to the present invention can be prepared byproteolytic hydrolysis of the antibody or by expression in E. coli ormammalian cells (e.g. Chinese hamster ovary cell culture or otherprotein expression systems) of DNA encoding the fragment. Antibodyfragments can be obtained by pepsin or papain digestion of wholeantibodies by conventional methods. For example, antibody fragments canbe produced by enzymatic cleavage of antibodies with pepsin to provide a5S fragment denoted F(ab′)2. This fragment can be further cleaved usinga thiol reducing agent, and optionally a blocking group for thesulfhydryl groups resulting from cleavage of disulfide linkages, toproduce 3.5S Fab′ monovalent fragments. Alternatively, an enzymaticcleavage using pepsin produces two monovalent Fab′ fragments and an Fcfragment directly. These methods are described, for example, byGoldenberg, U.S. Pat. Nos. 4,036,945 and 4,331,647, and referencescontained therein, which patents are hereby incorporated by reference intheir entirety. See also Porter, R. R. [Biochem. J. 73: 119-126 (1959)].Other methods of cleaving antibodies, such as separation of heavy chainsto form monovalent light-heavy chain fragments, further cleavage offragments, or other enzymatic, chemical, or genetic techniques may alsobe used, so long as the fragments bind to the antigen that is recognizedby the intact antibody.

Fv fragments comprise an association of VH and VL chains. Thisassociation may be noncovalent, as described in Inbar et al. [Proc. Nat.Acad. Sci. USA 69: 2659-62 (1972)]. Alternatively, the variable chainscan be linked by an intermolecular disulfide bond or cross-linked bychemicals such as gluteraldehyde. Preferably, the Fv fragments compriseVH and VL chains connected by a peptide linker. These single-chainantigen binding proteins (sFv) are prepared by constructing a structuralgene comprising DNA sequences encoding the VH and VL domains connectedby an oligonucleotide. The structural gene is inserted into anexpression vector, which is subsequently introduced into a host cellsuch as E. coli. The recombinant host cells synthesize a singlepolypeptide chain with a linker peptide bridging the two V domains.Methods for producing sFvs are described, for example, by Whitlow andFilpula, Methods 2: 97-105 (1991); Bird et al., Science 242:423-426(1988); Pack et al., Bio/Technology 11:1271-77 (1993); and U.S. Pat. No.4,946,778, which is hereby incorporated by reference in its entirety.

Another form of an antibody fragment is a peptide coding for a singlecomplementarity-determining region (CDR). CDR peptides (“minimalrecognition units”) can be obtained by constructing genes encoding theCDR of an antibody of interest. Such genes are prepared, for example, byusing the polymerase chain reaction to synthesize the variable regionfrom RNA of antibody-producing cells. See, for example, Larrick and Fry[Methods, 2: 106-10 (1991)].

Humanized forms of non-human (e.g., murine) antibodies are chimericmolecules of immunoglobulins, immunoglobulin chains or fragments thereof(such as Fv, Fab, Fab′, F(ab′).sub.2 or other antigen-bindingsubsequences of antibodies) which contain minimal sequence derived fromnon-human immunoglobulin. Humanized antibodies include humanimmunoglobulins (recipient antibody) in which residues form acomplementary determining region (CDR) of the recipient are replaced byresidues from a CDR of a non-human species (donor antibody) such asmouse, rat or rabbit having the desired specificity, affinity andcapacity. In some instances, Fv framework residues of the humanimmunoglobulin are replaced by corresponding non-human residues.Humanized antibodies may also comprise residues which are found neitherin the recipient antibody nor in the imported CDR or frameworksequences. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the CDR regions correspond to thoseof a non-human immunoglobulin and all or substantially all of the FRregions are those of a human immunoglobulin consensus sequence. Thehumanized antibody optimally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin [Jones et al., Nature, 321:522-525 (1986); Riechmann etal., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol.,2:593-596 (1992)].

Methods for humanizing non-human antibodies are well known in the art.Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues are often referred to as import residues, which aretypically taken from an import variable domain. Humanization can beessentially performed following the method of Winter and co-workers[Jones et al., Nature, 321: 522-525 (1986); Riechmann et al., Nature332: 323-327 (1988); Verhoeyen et al., Science, 239: 1534-1536 (1988)1,by substituting rodent CDRs or CDR sequences for the correspondingsequences of a human antibody. Accordingly, such humanized antibodiesare chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantiallyless than an intact human variable domain has been substituted by thecorresponding sequence from a non-human species. In practice, humanizedantibodies are typically human antibodies in which some CDR residues andpossibly some FR residues are substituted by residues from analogoussites in rodent antibodies.

Human antibodies can also be produced using various techniques known inthe art, including phage display libraries [Hoogenboom and Winter, J.Mol. Biol., 227: 381 (1991); Marks et al., J. Mol. Biol., 222: 581(1991)]. The techniques of Cole et al. and Boerner et al. are alsoavailable for the preparation of human monoclonal antibodies (Cole etal., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77(1985) and Boemer et al., J. Immunol., 147(1): 86-95 (1991)]. Similarly,human antibodies can be made by introduction of human immunoglobulinloci into transgenic animals, e.g., mice in which the endogenousimmunoglobulin genes have been partially or completely inactivated. Uponchallenge, human antibody production is observed, which closelyresembles that seen in humans in all respects, including generearrangement, assembly, and antibody repertoire. This approach isdescribed, for example, in U.S. Pat. Nos. 5,545,807; 5,545,806;5,569,825; 5,625,126; 5,633,425; 5,661,016, and in the followingscientific publications: Marks et al., Bio/Technology 10: 779-783(1992); Lonberg et al., Nature 368: 856-859 (1994); Morrison, Nature368: 812-13 (1994); Fishwild et al., Nature Biotechnology 14: 845-51(1996); Neuberger, Nature Biotechnology 14: 826 (1996); and Lonberg andHuszar, Intern. Rev. Immunol. 13: 65-93 (1995).

As is described below, various approaches can be used to reduce orabolish transcription or translation of a lysyl oxidase. These includeantisense oligonucleotides, Ribozyme, DNAzyme, siRNA and triple helixforming oligonucleotide approaches.

Antisense Polynucleotide

Downregulation of a lysyl oxidase can be effected using an antisensepolynucleotide capable of specifically hybridizing with an mRNAtranscript encoding the lysyl oxidase protein.

Design of antisense molecules which can be used to efficientlydownregulate lysyl oxidase must be effected while considering twoaspects important to the antisense approach. The first aspect isdelivery of the oligonucleotide into the cytoplasm of the appropriatecells, while the second aspect is design of an oligonucleotide whichspecifically binds the designated mRNA within cells in a way whichinhibits translation thereof

Several considerations must be taken into account when designingantisense oligonucleotides. For efficient in vivo inhibition of geneexpression using antisense oligonucleotides or analogs, theoligonucleotides or analogs must fulfill the following requirements (i)sufficient specificity in binding to the target sequence; (ii)solubility in water; (iii) stability against intra- and extracellularnucleases; (iv) capability of penetration through the cell membrane; and(v) when used to treat an organism, low toxicity. Algorithms foridentifying those sequences with the highest predicted binding affinityfor their target mRNA based on a thermodynamic cycle that accounts forthe energy of structural alterations in both the target mRNA and theoligonucleotide are also available [see, for example, Walton et al.Biotechnol Bioeng 65: 1-9 (1999)].

Such algorithms have been successfully used to implement an antisenseapproach in cells. For example, the algorithm developed by Walton et al.enabled scientists to successfully design antisense oligonucleotides forrabbit beta-globin (RBG) and mouse tumor necrosis factor-alpha (TNFalpha) transcripts. The same research group has more recently reportedthat the antisense activity of rationally selected oligonucleotidesagainst three model target mRNAs (human lactate dehydrogenase A and Band rat gp130) in cell culture as evaluated by a kinetic PCR techniqueproved effective in almost all cases, including tests against threedifferent targets in two cell types with phosphodiester andphosphorothioate oligonucleotide chemistries.

In addition, several approaches for designing and predicting efficiencyof specific oligonucleotides using an in vitro system were alsopublished (Matveeva et al., Nature Biotechnology 16: 1374-1375 (1998)].

An antisense molecule which can be used with the present inventionincludes a polynucleotide or a polynucleotide analog of at least 10bases, preferably between 10 and 15, more preferably between 15 and 20bases, most preferably at least 17, at least 18, at least 19, at least20, at least 22, at least 25, at least 30 or at least 40 bases which ishybridizable in vivo, under physiological conditions, with a portion ofa polynucleotide strand encoding a polypeptide at least 50% homologousto SEQ ID NO:1, 4, 5 or 7 or at least 75% homologous to an N-terminalportion thereof as determined using the BestFit software of theWisconsin sequence analysis package, utilizing the Smith and Watermanalgorithm, where gap creation penalty equals 8 and gap extension penaltyequals 2.

The antisense oligonucleotides used by the present invention can beexpressed from a nucleic acid construct administered into the tissue, inwhich case inducible promoters are preferably used such that antisenseexpression can be switched on and off, or alternatively sucholigonucleotides can be chemically synthesized and administered directlyinto the tissue, as part of, for example, a pharmaceutical composition.

The ability of chemically synthesizing oligonucleotides and analogsthereof having a selected predetermined sequence offers means fordownmodulating gene expression. Four types of gene expression modulationstrategies may be considered.

At the transcription level, antisense or sense oligonucleotides oranalogs that bind to the genomic DNA by strand displacement or theformation of a triple helix, may prevent transcription. At thetranscript level, antisense oligonucleotides or analogs that bind targetmRNA molecules lead to the enzymatic cleavage of the hybrid byintracellular RNase H. In this case, by hybridizing to the targetedmRNA, the oligonucleotides or oligonucleotide analogs provide a duplexhybrid recognized and destroyed by the RNase H enzyme. Alternatively,such hybrid formation may lead to interference with correct splicing. Asa result, in both cases, the number of the target mRNA intacttranscripts ready for translation is reduced or eliminated.

At the translation level, antisense oligonucleotides or analogs thatbind target mRNA molecules prevent, by steric hindrance, binding ofessential translation factors (ribosomes), to the target mRNA, aphenomenon known in the art as hybridization arrest, disabling thetranslation of such mRNAs.

Unmodified oligonucleotides are typically impractical for use asantisense sequences since they have short in vivo half-lives, duringwhich they are degraded rapidly by nucleases. Furthermore, they aredifficult to prepare in more than milligram quantities. In addition,such oligonucleotides are poor cell membrane penetrants.

Thus it is apparent that in order to meet all the above listedrequirements, oligonucleotide analogs need to be devised in a suitablemanner.

For example, problems arising in connection with double-stranded DNA(dsDNA) recognition through triple helix formation have been diminishedby a clever “switch back” chemical linking, whereby a sequence ofpolypurine on one strand is recognized, and by “switching back”, ahomopurine sequence on the other strand can be recognized. Also, goodhelix formation has been obtained by using artificial bases, therebyimproving binding conditions with regard to ionic strength and pH.

In addition, in order to improve half-life as well as membranepenetration, a large number of variations in polynucleotide backboneshave been done, nevertheless with little success.

Oligonucleotides can be modified either in the base, the sugar or thephosphate moiety. These modifications include, for example, the use ofmethylphosphonates, monothiophosphates, dithiophosphates,phosphoramidates, phosphate esters, bridged phosphorothioates, bridgedphosphoramidates, bridged methylenephosphonates, dephosphointemucleotide analogs with siloxane bridges, carbonate bridges,carboxymethyl ester bridges, carbonate bridges, carboxymethyl esterbridges, acetamide bridges, carbamate bridges, thioether bridges,sulfoxy bridges, sulfono bridges, various “plastic” DNAs, anomericbridges and borane derivatives [Cook (1991) Medicinal chemistry ofantisense oligonucleotides-future opportunities. Anti-Cancer Drug Design6: 585].

International patent application WO 89/12060 discloses various buildingblocks for synthesizing oligonucleotide analogs, as well asoligonucleotide analogs formed by joining such building blocks in adefined sequence. The building blocks may be either “rigid” (i.e.,containing a ring structure) or “flexible” (i.e., lacking a ringstructure). In both cases, the building blocks contain a hydroxy groupand a mercapto group, through which the building blocks are said to jointo form oligonucleotide analogs. The linking moiety in theoligonucleotide analogs is selected from the group consisting of sulfide(—S—), sulfoxide (—SO—), and sulfone (—SO2-).

International patent application WO 92/20702 describe an acyclicoligonucleotide which includes a peptide backbone on which any selectedchemical nucleobases or analogs are stringed and serve as codingcharacters as they do in natural DNA or RNA. These new compounds, knownas peptide nucleic acids (PNAs), are not only more stable in cells thantheir natural counterparts, but also bind natural DNA and RNA 50 to 100times more tightly than the natural nucleic acids cling to each other.PNA oligomers can be synthesized from the four protected monomerscontaining thymine, cytosine, adenine and guanine by Merrifieldsolid-phase peptide synthesis. In order to increase solubility in waterand to prevent aggregation, a lysine amide group is placed at theC-terminal region.

RNA oligonucleotides may also be used for antisense inhibition as theyform a stable RNA-RNA duplex with the target, suggesting efficientinhibition. However, due to their low stability RNA oligonucleotides aretypically expressed inside the cells using vectors designed for thispurpose. This approach is favored when attempting to target an mRNA thatencodes an abundant and long-lived protein.

The prior art teaches of a number of delivery strategies which can beused to efficiently deliver oligonucleotides into a wide variety of celltypes [see, for example, Luft J Mol Med 76: 75-6 (1998); Kronenwett etal. Blood 91: 852-62 (1998); Rajur et al. Bioconjug Chem 8: 935-40(1997); Lavigne et al. Biochem Biophys Res Commun 237: 566-71 (1997) andAoki et al. (1997) Biochem Biophys Res Commun 231: 540-5 (1997)].

Antisense therapeutics has the potential to treat many life-threateningdiseases with a number of advantages over traditional drugs. Traditionaldrugs intervene after a disease-causing protein is formed. Antisensetherapeutics, however, block mRNA transcription/translation andintervene before a protein is formed, and since antisense therapeuticstarget only one specific mRNA, they should be more effective with fewerside effects than current protein-inhibiting therapy.

Several clinical trials have demonstrated safety, feasibility andactivity of antisense oligonucleotides. For example, antisenseoligonucleotides suitable for the treatment of cancer have beensuccessfully used [Holmund et al., Curr Opin Mol Ther 1: 372-85 (1999)],while treatment of hematological malignancies via antisenseoligonucleotides targeting c-myb gene, p53 and Bcl-2 had enteredclinical trials and had been shown to be tolerated by patients [GerwitzCurr Opin Mol Ther 1: 297-306 (1999)].

More recently, antisense-mediated suppression of human heparanase geneexpression has been reported to inhibit pleural dissemination of humancancer cells in a mouse model [Uno et al., Cancer Res 61: 7855-60(2001)].

The first antisense drug was recently approved by the FDA. The drug,Fomivirsen, was developed by Isis, and is indicated for local treatmentof cytomegalovirus in patients with AIDS who are intolerant of or have acontraindication to other treatments for CMV retinitis or who wereinsufficiently responsive to previous treatments for CMV retinitis(Pharmacotherapy News Network).

Thus, the current consensus is that recent developments in the field ofantisense technology which, as described above, have led to thegeneration of highly accurate antisense design algorithms and a widevariety of oligonucleotide delivery systems, enable an ordinarilyskilled artisan to design and implement antisense approaches suitablefor downregulating expression of known sequences without having toresort to undue trial and error experimentation.

Ribozyme

Another agent capable of downregulating a lysyl oxidase is a ribozymemolecule capable of specifically cleaving an mRNA transcript encoding alysyl oxidase. Ribozymes are being increasingly used for thesequence-specific inhibition of gene expression by the cleavage of mRNAsencoding proteins of interest [Welch et al., Curr Opin Biotechnol. 9:486-96 (1998)]. The possibility of designing ribozymes to cleave anyspecific target RNA has rendered them valuable tools in both basicresearch and therapeutic applications. In the therapeutics area,ribozymes have been exploited to target viral RNAs in infectiousdiseases, dominant oncogenes in cancers and specific somatic mutationsin genetic disorders [Welch et al., Clin Diagn Virol. 10: 163-71(1998)]. Most notably, several ribozyme gene therapy protocols for HIVpatients are already in Phase 1 trials. More recently, ribozymes havebeen used for transgenic animal research, gene target validation andpathway elucidation. Several ribozymes are in various stages of clinicaltrials. ANGIOZYME was the first chemically synthesized ribozyme to bestudied in human clinical trials. ANGIOZYME specifically inhibitsformation of the VEGF-r (Vascular Endothelial Growth Factor receptor), akey component in the angiogenesis pathway. Ribozyme Pharmaceuticals,Inc., as well as other firms have demonstrated the importance ofanti-angiogenesis therapeutics in animal models. HEPTAZYME, a ribozymedesigned to selectively destroy Hepatitis C Virus (HCV) RNA, was foundeffective in decreasing Hepatitis C viral RNA in cell culture assays(Ribozyme Pharmaceuticals, Incorporated—WEB home page).

DNAzyme

Another agent capable of downregulating a lysyl oxidase is a DNAzymemolecule capable of specifically cleaving an mRNA transcript or DNAsequence of the lysyl oxidase. DNAzymes are single-strandedpolynucleotides which are capable of cleaving both single and doublestranded target sequences (Breaker, R. R. and Joyce, G. Chemistry andBiology 1995; 2: 655; Santoro, S. W. & Joyce, G. F. Proc. Natl, Acad.Sci. USA 1997; 943: 4262). A general model (the “10-23” model) for theDNAzyme has been proposed. “10-23” DNAzymes have a catalytic domain of15 deoxyribonucleotides, flanked by two substrate-recognition domains ofseven to nine deoxyribonucleotides each. This type of DNAzyme caneffectively cleave its substrate RNA at purine:pyrimidine junctions(Santoro, S. W. & Joyce, G. F. Proc. Natl, Acad. Sci. USA 1997; 943:4262; for rev of DNAzymes see Khachigian, L M [Curr Opin Mol Ther 4:119-21 (2002)].

Examples of construction and amplification of synthetic, engineeredDNAzymes recognizing single and double-stranded target cleavage siteshave been disclosed in U.S. Pat. No. 6,326,174 to Joyce et al. DNAzymesof similar design directed against the human Urokinase receptor wererecently observed to inhibit Urokinase receptor expression, andsuccessfully inhibit colon cancer cell metastasis in vivo (Itoh et al.,2002, Abstract 409, Ann Meeting Am. Soc. Gen. Ther., located on theWorld Wide Web at asgt.org). In another application, DNAzymescomplementary to bcr-abl oncogenes were successful in inhibiting theoncogenes expression in leukemia cells, and lessening relapse rates inautologous bone marrow transplant in cases of CML and ALL.

siRNA

Another mechanism of down regulating a lysyl oxidase at the transcriptlevel is RNA interference (RNAi), an approach which utilizes smallinterfering dsRNA (siRNA) molecules that are homologous to the targetmRNA and lead to its degradation [Carthew R W. Gene silencing bydouble-stranded RNA. Curr Opin Cell Biol 2001 April; 13(2):244-8].

RNA interference is a two-step process. In the first step, which istermed as the initiation step, input dsRNA is digested into 21-23nucleotide (nt) small interfering RNAs (siRNA), probably by the actionof Dicer, a member of the RNase III family of dsRNA-specificribonucleases, which processes (cleaves) dsRNA (introduced directly orvia a transgene or a virus) in an ATP-dependent manner. Successivecleavage events degrade the RNA to 19-21 bp duplexes (siRNA), each with2-nucleotide 3′ overhangs [Hutvagner and Zamore Curr. Opin. Genetics andDevelopment 12: 225-232 (2002); and Bernstein Nature 409: 363-366(2001)].

In the effector step, the siRNA duplexes bind to a nuclease complex toform the RNA-induced silencing complex (RISC). An ATP-dependentunwinding of the siRNA duplex is required for activation of the RISC.The active RISC then targets the homologous transcript by base pairinginteractions and cleaves the mRNA into 12 nucleotide fragments from the3′ terminus of the siRNA [Hutvagner and Zamore Curr. Opin. Genetics andDevelopment 12: 225-232 (2002); Hammond et al. (2001) Nat. Rev. Gen. 2:110-119 (2001); and Sharp Genes. Dev. 15: 485-90 (2001)]. Although themechanism of cleavage is still to be elucidated, research indicates thateach RISC contains a single siRNA and an RNase [Hutvagner and ZamoreCurr. Opin. Genetics and Development 12: 225-232 (2002)].

Because of the remarkable potency of RNAi, an amplification step withinthe RNAi pathway has been suggested. Amplification could occur bycopying of the input dsRNAs which would generate more siRNAs, or byreplication of the siRNAs formed. Alternatively or additionally,amplification could be effected by multiple turnover events of the RISC[Hammond et al. Nat. Rev. Gen. 2: 110-119 (2001), Sharp Genes. Dev. 15:485-90 (2001); Hutvagner and Zamore Curr. Opin. Genetics and Development12: 225-232 (2002)]. For more information on RNAi see the followingreviews Tuschl Chem Biochem. 2: 239-245 (2001); Cullen Nat. Immunol. 3:597-599 (2002); and Brantl Biochem. Biophys. Act. 1575: 15-25 (2002).

Synthesis of RNAi molecules suitable for use with the present inventioncan be effected as follows. First, the lysyl oxidase mRNA sequence isscanned downstream of the AUG start codon for AA dinucleotide sequences.Occurrence of each AA and the 3′ adjacent 19 nucleotides is recorded aspotential siRNA target sites. Preferably, siRNA target sites areselected from the open reading frame, as untranslated regions (UTRs) arericher in regulatory protein binding sites. UTR-binding proteins and/ortranslation initiation complexes may interfere with binding of the siRNAendonuclease complex [Tuschl Chem Biochem. 2: 239-245, 2001]. It will beappreciated though, that siRNAs directed at untranslated regions mayalso be effective, as demonstrated for GAPDH wherein siRNA directed atthe 5′ UTR mediated about 90% decrease in cellular GAPDH mRNA andcompletely abolished protein level (located on the World Wide Web atambion.com/techlib/tn/91/912.html).

Second, potential target sites are compared to an appropriate genomicdatabase (e.g., human, mouse, rat etc.) using any sequence alignmentsoftware, such as the BLAST software available from the NCBI server(located on the World Wide Web at ncbi.nlm.nih.gov/BLAST/). Putativetarget sites which exhibit significant homology to other codingsequences are filtered out.

Qualifying target sequences are selected as template for siRNAsynthesis. Preferred sequences are those including low G/C content asthese have proven to be more effective in mediating gene silencing ascompared to those with G/C content higher than 55%. Several target sitesare preferably selected along the length of the target gene forevaluation. For better evaluation of the selected siRNAs, a negativecontrol is preferably used in conjunction. Negative control siRNApreferably include the same nucleotide composition as the siRNAs butlack significant homology to the genome. Thus, a scrambled nucleotidesequence of the siRNA is preferably used, provided it does not displayany significant homology to any other gene.

The siRNA molecules of the present invention are preferably transcribedfrom expression vectors which can facilitate stable expression of thesiRNA transcripts once introduced into a host cell. These vectors areengineered to express small hairpin RNAs (shRNAs), which are processedin vivo into siRNA molecules capable of carrying out gene-specificsilencing [Brummelkamp, T. R., et al., (2002) A system for stableexpression of short interfering RNAs in mammalian cells. Science 296:550-53; Paddison, P. J., et al. (2002) Short hairpin RNAs (shRNAs)induce sequence-specific silencing in mammalian cells. Genes Dev.16:948-58; Paul et al. (2002) Nature Biotech. 20: 505-08; Yu, J. Y., etal. (2002) RNA interference by expression of short-interfering RNAs andhairpin RNAs in mammalian cells. Proc. Natl. Acad. Sci. USA 99: 6047-52]

An example of a suitable expression vector is the pSUPER™, whichincludes the polymerase-III H1-RNA gene promoter with a well definedstart of transcription and a termination signal consisting of fivethymidines in a row (T5) [Brummelkamp, T. R. et al. (2002), Science 296:550-53]. Most importantly, the cleavage of the transcript at thetermination site is at a site following the second uridine, thusyielding a transcript which resembles the ends of synthetic siRNAs,which also contain nucleotide overhangs. siRNA is cloned such that itincludes the sequence of interest, i.e., lysyl oxidase separated by ashort spacer from the reverse complement of the same sequence. Theresulting transcript folds back on itself to form a stem-loop structure,which mediates lysyl oxidase RNAi.

Another suitable siRNA expression vector encodes the sense and antisensesiRNA under the regulation of separate polIII promoters [Miyagishi andTaira (2002) Nature Biotech. 20: 497-500]. The siRNA, generated by thisvector also includes a five thymidine (T5) termination signal.

Since approaches for introducing synthetic siRNA into cells bylipofection can result in low transfection efficiencies in some celltypes and/or short-term persistence of silencing effects, vectormediated methods have been developed.

Thus, siRNA molecules utilized by the present invention are preferablydelivered into cell using retroviruses. Delivery of siRNA usingretroviruses provides several advantages over methods, such aslipofection, since retroviral delivery is more efficient, uniform andimmediately selects for stable “knock-down” cells [Devroe, E. andSilver, P. A. (2002). Retrovirus-delivered siRNA. BMC Biotechnol. 2: 15]

Recent scientific publications have validated the efficacy of such shortdouble stranded RNA molecules in inhibiting target mRNA expression andthus have clearly demonstrated the therapeutic potential of suchmolecules. For example, RNAi has been utilized to inhibit expression ofhepatitis C (McCaffrey, A. P., et al., 2002, Gene expression: RNAinterference in adult mice. Nature 418, 38-39), HIV-1 (Jacque, J-M., etal. 2002, Modulation of HIV-1 replication by RNA interference. Nature418, 435-438), cervical cancer cells (Jiang, M., and Milner, J. 2002,Selective silencing of viral gene expression in HPV-positive humancervical carcinoma cells treated with siRNA, a primer of RNAinterference. Oncogene 21, 6041-8) and leukemic cells [Wilda, M., etal., 2002, Killing of leukemic cells with a BCR/ABL fusion gene by RNAinterference (RNAi). Oncogene 21, 5716-24].

Triple Helix Forming Oligonucleotide (TFO)

An additional method of regulating the expression of a lysyl oxidase incells is via triplex forming oligonucleotides (TFOs). Recent studieshave shown that TFOs can be designed which can recognize and bind topolypurine/polypyrimidine regions in double-stranded helical DNA in asequence-specific manner. These recognition rules are outlined by MaherIII, L. J., et al., Science (1989), 245: 725-730; Moser, H. E., et al.,Science (1987), 238: 645-630; Beal, P. A., et al, Science (1992), 251:1360-1363; Cooney, M., et al., Science (1988), 241: 456-459; and Hogan,M. E., et al., EP Publication 375408. Modification of theoligonucleotides, such as the introduction of intercalators and backbonesubstitutions, and optimization of binding conditions (pH and cationconcentration) have aided in overcoming inherent obstacles to TFOactivity such as charge repulsion and instability, and it was recentlyshown that synthetic oligonucleotides can be targeted to specificsequences [for a recent review see Seidman and Glazer, J. Clin. Invest.(2003), 112: 487-94].

In general, the triplex-forming oligonucleotide has the sequencecorrespondence:

oligo 3′-AGGT duplex 5′-AGCT duplex 3′-TCGA

However, it has been shown that the A-AT and G-GC triplets have thegreatest triple helical stability (Reither and Jeltsch, BMC Biochem,2002, Sep. 12, Epub). The same authors have demonstrated that TFOsdesigned according to the A-AT and G-GC rule do not form non-specifictriplexes, indicating that the triplex formation is indeed sequencespecific.

Thus for any given sequence in the lysyl oxidase regulatory region atriplex forming sequence may be devised. Triplex-formingoligonucleotides preferably are at least 15, more preferably 25, stillmore preferably 30 or more nucleotides in length, up to 50 or 100 bp.

Transfection of cells (for example, via cationic liposomes) with TFOs,and formation of the triple helical structure with the target DNAinduces steric and functional changes, blocking transcription initiationand elongation, allowing the introduction of desired sequence changes inthe endogenous DNA and resulting in the specific downregulation of geneexpression. Examples of such suppression of gene expression in cellstreated with TFOs include knockout of episomal supFG1 and endogenousHPRT genes in mammalian cells (Vasquez et al., Nucl Acids Res. 1999; 27:1176-81, and Puri, et al, J Biol Chem, 2001; 276: 28991-98), and thesequence- and target specific downregulation of expression of the Ets2transcription factor, important in prostate cancer etiology (Carbone, etal, Nucl Acid Res. 2003; 31: 833-43), and the pro-inflammatory ICAM-1gene (Besch et al, J Biol Chem, 2002; 277: 32473-79). In addition,Vuyisich and Beal have recently shown that sequence specific TFOs canbind to dsRNA, inhibiting activity of dsRNA-dependent enzymes such asRNA-dependent kinases (Vuyisich and Beal, Nuc. Acids Res 2000; 28:2369-74).

Additionally, TFOs designed according to the abovementioned principlescan induce directed mutagenesis capable of effecting DNA repair, thusproviding both downregulation and upregulation of expression ofendogenous genes (Seidman and Glazer, J Clin Invest 2003; 112: 487-94).Detailed description of the design, synthesis and administration ofeffective TFOs can be found in U.S. Pat. Appl. Nos. 2003 017068 and 20030096980 to Froehler et al, and 2002 0128218 and 2002 0123476 to Emanueleet al, and U.S. Pat. No. 5,721,138 to Lawn.

The downregulators described hereinabove would be particularly usefulfor inhibiting angiogenesis in tumor tissue. It has been shown that PF4,a lysyl oxidase binding protein which inhibits angiogenesis in tumortissue specifically accumulates in newly formed blood vessels of tumors(angiogenic vessels) but not in established blood vessels (Hansell, P.,et al., 1995, Selective binding of platelet factor 4 to regions ofactive angiogenesis in vivo. Amer. J. Physiol-Heart. Circ. Phy. 38,H829-H836; Reiser, K., et al., 1992, Enzymatic and nonenzymaticcross-linking of collagen and elastin. FASEB J. 6, 2439-2449).

Newly formed angiogenic blood vessels are more permeable to proteinsthan established blood vessels because the major inducer of angiogenesisin many angiogenic diseases is VEGF, a growth factor which alsofunctions as a potent blood vessel permeabilizing factor (VPF) [Neufeldet al., 1999, Vascular endothelial growth factor (VEGF) and itsreceptors. FASEB J. 13(1): 9-22]. Tumor associated blood vessels aretherefore in a permanent state of hyperpermeability due to deregulatedover-expression of VEGF (Shweiki et al., 1995; Rak et al., 1995) and assuch, a downregulator molecule used by the method of the presentinvention would be able to extravasate efficiently from tumor bloodvessels but much less efficiently from normal stabilized blood vessels.

Upregulators

Several approaches can be utilized to increase the levels of lysyloxidase and as such to enhance the formation of blood vessels.

For example, a nucleic acid construct including a constitutive,inducible or tissue specific promoter positioned upstream of apolynucleotide encoding a polypeptide having lysyl oxidase activity,such as the polypeptide set forth in SEQ ID NO:2, 3, 6, 8 or 9 can beadministered into a mammalian tissue. The lysyl oxidase expressed fromthis construct would substantially increase the levels of lysyl oxidasewithin the cells of the tissue and as such enhance angiogenesis.

The polynucleotide segments encoding the lysyl oxidase can be ligatedinto a commercially available expression vector. Such an expressionvector includes a promoter sequence for directing transcription of thepolynucleotide sequence in the cell in a constitutive or induciblemanner. A suitable promoter can be, for example, a Tie-2 promoter whichis capable of directing lysyl oxidase specific gene expression inendothelial cells (see Schlaeger, T. M., Bartunkova, S., Lawitts, J. A.,Teichmann, G., Risau, W., Deutsch, U., and Sato, T. N. (1997). Uniformvascular-endothelial-cell-specific gene expression in both embryonic andadult transgenic mice. Proc. Natl. Acad. Sci. U.S.A 94, 3058-3063). Theexpression vector of the present invention can further includeadditional polynucleotide sequences such as for example, sequencesencoding selection markers or reporter polypeptides, sequences encodingorigin of replication in bacteria, sequences that allow for translationof several proteins from a single mRNA such as an internal ribosomeentry site (IRES), sequences for genomic integration of thepromoter-chimeric polypeptide encoding region and/or sequences generallyincluded in mammalian expression vector such as pcDNA3, pcDNA3.1(+/−),pZeoSV2(+/−), pSecTag2, pDisplay, pEF/myc/cyto, pCMV/myc/cyto, pCR3.1,which are available from Invitrogen, pCI which is available fromPromega, pBK-RSV and pBK-CMV which are available from Strategene, pTRESwhich is available from Clontech, and their derivatives.

It will be appreciated that such commercially available vector systemscan easily be modified via commonly used recombinant techniques in orderto replace, duplicate or mutate existing promoter or enhancer sequencesand/or introduce any additional polynucleotide sequences.

An agent capable of upregulating a lysyl oxidase may also be anycompound which is capable of increasing the transcription and/ortranslation of an endogenous DNA or mRNA encoding a lysyl oxidase usingfor example gene “knock in” techniques.

Enhancer elements can be “knocked-in” adjacent to endogenous lysyloxidase coding sequences to thereby increase transcription therefrom.

Further details relating to the construction and use of knock-out andknock-in constructs is provided elsewhere [Fukushige S. and Ikeda, J. E.Trapping of mammalian promoters by Cre-lox site-specific recombination.DNA Res 3 (1996) 73-80; Bedell, M. A., et al. Mouse models of humandisease. Part I: Techniques and resources for genetic analysis in mice.Genes and Development 11 (1997) 1-11; Bermingham, J. J., et al.Tst-1/Oct-6/SCIP regulates a unique step in peripheral myelination andis required for normal respiration. Genes Dev 10 (1996) 1751-62].

It will be appreciated that direct administration of a polypeptideexhibiting a lysyl oxidase activity can also be utilized for enhancingangiogenesis.

Thus, affinity binding assays and/or activity assays, the principles ofwhich are well known in the art, can be used to screen for novelcompounds (e.g., substrate analogs) which can specifically regulate theactivity of a lysyl oxidase and as such can be used with the presentinvention.

An assay suitable for use with this aspect of the present invention hasbeen previously described in a study conducted by Bedell-Hogan et al.,1993.

As is clearly illustrated in the Examples section which follows, thepresent study also correlated expression levels of LOR-1 to themetastatic properties of breast cancer derived cell lines, indicatingthat LOR-1 may play additional roles in tumor invasiveness in additionto its role in angiogenesis.

Thus, the present invention also provides a method of inhibitingmetastasis and/or fibrosis in a mammalian tissue. The method is effectedby administering to the mammalian tissue a molecule capable ofdownregulating a tissue level and/or an activity of at least one type ofa lysyl oxidase.

The method of the present invention can be used to treat human patientsthat have been diagnosed with cancerous tumors, by administering any ofthe downregulating molecules described herein above, in order to reducethe tissue level and/or activity of at least one type of a lysyloxidase.

As used herein, the phrase “cancerous tumor” refers to any malignanttumor within a human body including, but not limiting to, tumors withmetastases. In addition, and without being bound to any particular typeof cancerous tumor, the present invention is useful to treat breastcancer tumors, with or without metastases.

As used herein, the phrase “administering” refers to all modes ofadministration described hereinbelow with respect to the pharmaceuticalcompositions of the present invention.

These include, but not limit to, local administration at the tumortissue, an organ where the cancerous tumor was diagnosed and/or relatedtissues that typically form metastases [Hortobagyi, 2002, Semin Oncol 29(3 Suppl 11): 134-44; Morrow and Gradishar, 2002, 324: 410-4]. Examplesof related tissue include lymph nodes adjacent to, for example, breasttissue and bones.

Administration can also be effected in a systemic manner in order totreat the affected tissue, i.e., the tissue where the cancerous tumorwas formed and where metastases are present or likely to be formed withtumor progression.

Since any molecule capable of downregulating a lysyl oxidase activitycan be utilized by the methods described hereinabove, the presentinvention also provides a method of identifying molecules capable ofinhibiting metastasis and/or fibrosis.

This method is effected by screening and identifying molecules whichexhibit specific reactivity with at least one type of lysyl oxidase andtesting a metastasis and/or fibrosis inhibitory potential of thesemolecules.

Numerous types of molecules can be screened for reactivity with at leastone type of lysyl oxidase, examples include, but are not limited to,molecules such as antisense oligonucleotides, siRNA, DNAzymes, ribozymesand triple helix forming oligonucleotides (TFOs) that interact with apolynucleotide expressing a lysyl oxidase activity or molecules such asantibodies that interact with polypeptides having a lysyl oxidaseactivity. In addition, short peptides and other small molecules can alsobe screened by this method of the present invention.

Screening for cross reactivity can be effected by lysyl oxidaseenzymatic activity assays, by binding assays and the like. Examples ofsuitable assays are provided in Rodriguez et al., 2002, ArteriosclerThromb Vasc Biol 22: 1409-14; Wilson and Nock, 2002, Curr Opin Chem Biol6: 81-5; Uetz, 2002, Curr Opin Chem Biol 6: 57-62; Stoll et al., 2002,Front Biosci 2002 7: c13-32).

Testing a metastatic phenotype of transformed tumor cells can beperformed in vitro since nearly all steps of the metastatic process,including attachment, matrix degradation and migration, can be modeledexperimentally in vitro by measuring invasion of a reconstitutedbasement membrane (RBM). Metastatic invasiveness of tumor cell can bemodeled by migration of tumor cells into reconstituted basement membrane(RBM) in the presence and absence of a chemoattractant, such asfibroblast conditioned medium (FCM). The assay determines cells thathave attached to the RBM, degraded the RBM enzymatically and, finally,cells that have penetrated the FCM side of the membrane.

Since in vitro metastasis events correspond to steps observed in themetastatic spread of tumor cells through the basement membrane in vivo,in vitro invasiveness of cells can be assayed by the methods describedin Albini et al., 1987 Cancer Research 47: 3239-3245, which isincorporated herein by reference in its entirety. Invasiveness assaysand other methods for assessing metastatic affects, are described inLeyton et al., 1994 Cancer Research 54: 3696-3699, which is incorporatedby reference herein in its entirety. Reconstituted basement membranepreparations for use in accordance with the hereinabove described assaysare readily available from numerous commercial suppliers. One suitableexample membrane in this regard is “MATRIGEL” available fromCollaborative Biomedical Products of Bedford, Mass.

In vitro evaluation of tumor cell metastatic phenotype can also beeffected by determining level and pattern of expression of one or moremetastasis associated markers such protease markers, which areconsidered to be an integral part of tumor metastasis (see U.S. Pat. No.6,303,318). One example is the arachidonic acid, the release of which incells can serve to indicate metastatic potential of a tumor (U.S. Pat.No. 6,316,416). In this regard, determining phospholipase A-2 (PLA2)activity, and the activity or abundance of factors that affect theactivity of PLA2, such as uteroglobin protein (U.S. Pat. No. 6,316,416)can serve as an indication of metastatic potential.

Determining pattern and level of expression of metastasis-associatedmarkers can be effected by one of several methods known in the art.

The presence or level of proteins indicative of metastatic potential oftumors can be determined in cells by conventional methods well known tothose of skill in the art. For instance, the techniques for making andusing antibody and other immunological reagents and for detectingparticular proteins in samples using such reagents are described incurrent protocols in immunology, Coligan et al., Eds., John Wiley &Sons, New York (1995), which is incorporated by reference herein inparts pertinent to making and using reagents useful for determiningspecific proteins in samples. As another example, immunohistochemicalmethods for determining proteins in cells in tissues are described inVolume 2, Chapter 14 of current protocols in molecular biology, Ausubelet al., Eds., John Wiley & Sons, Inc. (1994), which is incorporated byreference herein in part pertinent to carrying out such determinations.Finally, Linnoila et al., A.J.C.P. 97(2): 235-243 (1992) and Peri etal., J. Clin. Invest. 92: 2099-2109 (1992), incorporated herein asreferred to above, describe techniques that may need, in part, in thisaspect of the present invention.

Metastatic potential can also be determined in vivo at the mRNA level.The presence and/or level of mRNA transcripts can be determined by avariety of methods known to those of skill in the art. A given mRNA maybe detected in cells by hybridization to a specific probe. Such probesmay be cloned DNAs or fragments thereof, RNA, typically made by in vitrotranscription, or oligonucleotide probes, usually generated by solidphase synthesis. Methods for generating and using probes suitable forspecific hybridization are well known and used in the art.

A variety of controls may be usefully employed to improve accuracy inmRNA detection assays. For instance, samples may be hybridized to anirrelevant probe and treated with RNAse A prior to hybridization, toassess false hybridization.

In order to modulate angiogenesis or inhibit metastasis or tumorfibrosis, the molecules used by the present invention can beadministered to the individual per se, or in a pharmaceuticalcomposition where it is mixed with suitable carriers or excipients.

As used herein a “pharmaceutical composition” refers to a preparation ofone or more of the active ingredients described herein with otherchemical components such as physiologically suitable carriers andexcipients. The purpose of a pharmaceutical composition is to facilitateadministration/targeting of a compound to a mammal.

As used herein the term “active ingredients”, refers to the preparationaccountable for the biological effect, i.e. theupregulator/downregulator molecules used by the present invention tomodulate angiogenesis and the downregulators molecules used by thepresent invention to inhibit metastasis and tumor fibrosis.

Hereinafter, the phrases “physiologically acceptable carrier” and“pharmaceutically acceptable carrier” are interchangeably used to referto a carrier, such as, for example, a liposome, a virus, a micelle, or aprotein, or a diluent which do not cause significant irritation to themammal and do not abrogate the biological activity and properties of theactive ingredient. An adjuvant is included under these phrases.

Herein the term “excipient” refers to an inert substance added to apharmaceutical composition to further facilitate administration of anactive ingredient. Examples, without limitation, of excipients, includecalcium carbonate, calcium phosphate, various sugars and types ofstarch, cellulose derivatives, gelatin, vegetable oils and polyethyleneglycols.

Techniques for formulation and administration of compositions may befound in “Remington's Pharmaceutical Sciences” Mack Publishing Co.,Easton, Pa., latest edition, which is incorporated herein by reference.

Suitable routes of administration may, for example, include oral,rectal, transmucosal, transnasal, intestinal or parenteral delivery,including intramuscular, subcutaneous and intramedullary injections aswell as intrathecal, direct intraventricular, intravenous,inrtaperitoneal, intranasal, or intraocular injections.

For injection, the active ingredients of the invention may be formulatedin aqueous solutions, preferably in physiologically compatible bufferssuch as Hank's solution, Ringer's solution, or physiological saltbuffer. For transmucosal administration, penetrants appropriate to thebarrier to be permeated are used in the formulation. Such penetrants aregenerally known in the art.

For oral administration, the compounds can be formulated readily bycombining the active ingredient with pharmaceutically acceptablecarriers well known in the art. Such carriers enable the activeingredient of the invention to be formulated as tablets, pills, dragees,capsules, liquids, gels, syrups, slurries, suspensions, and the like,for oral ingestion by a patient. Pharmacological preparations for oraluse can be made using a solid excipient, optionally grinding theresulting mixture, and processing the mixture of granules, after addingsuitable auxiliaries if desired, to obtain tablets or dragee cores.Suitable excipients are, in particular, fillers such as sugars,including lactose, sucrose, mannitol, or sorbitol; cellulosepreparations such as, for example, maize starch, wheat starch, ricestarch, potato starch, gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl-cellulose, sodium carbomethylcellulose; and/orphysiologically acceptable polymers such as polyvinylpyrrolidone (PVP).If desired, disintegrating agents may be added, such as cross-linkedpolyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such assodium alginate.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used which may optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, titanium dioxide, lacquer solutions and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active compound doses.

Pharmaceutical compositions, which can be used orally, include push-fitcapsules made of gelatin as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules may contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, lubricants such as talc ormagnesium stearate and, optionally, stabilizers. In soft capsules, theactive ingredients may be dissolved or suspended in suitable liquids,such as fatty oils, liquid paraffin, or liquid polyethylene glycols. Inaddition, stabilizers may be added. All formulations for oraladministration should be in dosages suitable for the chosen route ofadministration.

For buccal administration, the compositions may take the form of tabletsor lozenges formulated in conventional manner.

The preparations described herein may be formulated for parenteraladministration, e.g., by bolus injection or continuous infusion.Formulations for injection may be presented in unit dosage form, e.g.,in ampoules or in multidose containers with optionally, an addedpreservative. The compositions may be suspensions, solutions oremulsions in oily or aqueous vehicles, and may contain formulatoryagents such as suspending, stabilizing and/or dispersing agents.

Pharmaceutical compositions for parenteral administration includeaqueous solutions of the active preparation in water-soluble form.Additionally, suspensions of the active ingredients may be prepared asappropriate oily or water based injection suspensions. Suitablelipophilic solvents or vehicles include fatty oils such as sesame oil,or synthetic fatty acids esters such as ethyl oleate, triglycerides orliposomes. Aqueous injection suspensions may contain substances, whichincrease the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol or dextran. Optionally, the suspension may alsocontain suitable stabilizers or agents which increase the solubility ofthe active ingredients to allow for the preparation of highlyconcentrated solutions.

Alternatively, the active ingredient may be in powder form forconstitution with a suitable vehicle, e.g., sterile, pyrogen-free waterbased solution, before use.

The preparation of the present invention may also be formulated inrectal compositions such as suppositories or retention enemas, using,e.g., conventional suppository bases such as cocoa butter or otherglycerides.

Pharmaceutical compositions suitable for use in context of the presentinvention include compositions wherein the active ingredients arecontained in an amount effective to achieve the intended purpose.

The pharmaceutical composition may form a part of an article ofmanufacturing which also includes a packaging material for containingthe pharmaceutical composition and a leaflet which provides indicationsof use for the pharmaceutical composition.

Thus, the present invention provides a method and pharmaceuticalcompositions useful modulating angiogenesis.

Such modulation activity can be used to treat arthritis (Koch, 1998;Paleolog and Fava, 1998), diabetic retinopathy (Miller et al., 1997),psoriasis (Detmar et al., 1994; Creamer et al., 1997) and vasculitis(Lie, 1992; Klipple and Riordan, 1989).

In addition, the present invention can also be used to treat diseasecharacterized by fragile blood vessels, including Marfans syndrome,Kawasaki, Ehlers-Danlos, cutis-laxa, and takysu (Lie, 1992; Klipple andRiordan, 1989; Brahn et al., 1999; Cid et al., 1993; Hoffman et al.,1991).

It is possible that some of these diseases result from reduced orabolished lysyl oxidase activity which leads to the synthesis of afragile extracellular matrix, and consequently, fragile blood vessels.

As such, administration of lysyl oxidase encoding sequences orpolypeptides can be used to correct some of the manifestations of thesediseases.

The present invention can also be used to treat diseases which arecharacterized by changes in the wall of blood vessels. For example,restenosis which is a common complication following balloon therapy,Fibromuscular dysplasia (Begelman and Olin, 2000) and aortic stenosis(Palta et al., 2000) are all potentially treatable by the method of thepresent invention.

In addition, as is illustrated in the Examples section which follows,LOR-1 is more highly expressed in metastatic tumors and cell lines thanin non-metastatic tumors and cell lines (FIGS. 3, 9, and 13). Thissuggests that levels of LOR-1 expression can be used as a diagnostictool to determine the malignancy of cancer cells, as well as, todetermine and implement suitable treatment regimens.

Colon cancer is a highly treatable and often a curable disease whenlocalized to the bowel. However, in many cases, due to mis-diagnosis, apre-malignant colon hyperplasia progress into colon adenoma whichfurther develop into more malignant forms of low-grade and high-gradecolon adenocarcinoma. Once an individual is diagnosed with colon cancerthe malignancy of the tumor needs to be assessed in order to select forsuitable treatment regimens. The current practice for assessing themalignancy of a colon tumor is based on the tumor-node-metastases (TNM)staging system developed by the American Joint Committee on Cancer(AJCC). According to this method staging is based on scoring for thepresence or absence of cancerous cells in the tumor itself, in thesubmucosa of the bowel wall, in the muscular layer of the bowel wall(muscularis propria), and/or in the subserosa, pericolic or perirectaltissues, as well as in regional lymph nodes and distance metastases.Thus, staging of colon tumors involves multiple tissue biopsies andcomplex pathological evaluations which are time consuming and can resultin misdiagnosis.

While reducing the present invention to practice, the present inventorshave also uncovered that LOR-1 expression in epithelial and/orconnective tissue cells in a colon tissue is indicative of a malignantcolon cancer thus providing a new method of assessing a malignancy ofcolon cancer tumors devoid of the above limitations.

As described in Example 3 of the Examples section which follows, theexpression of LOR-1 is correlated with the formation of benign colontumors (FIG. 14b ) and is increased in more malignant forms of coloncancer tumors (FIGS. 14c and 14d ) thus suggesting the use of LOR-1 indetermining the stage of colon cancer tumors.

Thus according to another aspect of the present invention there isprovided a method of assessing a malignancy of a colon tumor. The methodis effected by determining a tissue level and/or an activity level of apolypeptide at least 75% homologous to the polypeptide set forth in SEQID NO:2 or 9 in the colon tumor tissue, thereby assessing the malignancyof the colon tumor.

As is used herein, the phrase “assessing a malignancy of a colon tumor”refers to determining the stage of the colon tumor, i.e., the progressof the colon tumor from a benign colon tumor to a highly malignant coloncancer which invades the surrounding tissue.

The polypeptide detected by the present invention is at least 75%, atleast 80%, at least 85%, at least 90%, at least 95% homologous to SEQ IDNO:2 or 9, as determined using the BestFit software of the Wisconsinsequence analysis package, utilizing the Smith and Waterman algorithm,where gap creation penalty equals 8 and gap extension penalty equals 2.

Preferably, the polypeptide of the present invention is LOR-1 (SEQ IDNO:2), a member of the lysyl oxidase family which are fully describedherein above.

According to the method of the present invention, a colon tumor tissueis obtained using a colon biopsy and/or a colon surgery using methodsknow in the art. Once obtained, the tissue level and/or activity levelof the polypeptide of the present invention is determined in the colontumor tissue.

Determination of the tissue level of the polypeptide of the presentinvention may be accomplished directly using immunological methods.

The immunological detection methods used in context of the presentinvention are fully explained in, for example, “Using Antibodies: ALaboratory Manual” (Ed Harlow, David Lane eds., Cold Spring HarborLaboratory Press (1999)) and those familiar with the art will be capableof implementing the various techniques summarized hereinbelow as part ofthe present invention. All of the immunological techniques requireantibodies specific to at least one epitope of the polypeptide of thepresent invention. Immunological detection methods suited for use aspart of the present invention include, but are not limited to,radio-immunoassay (RIA), enzyme linked immunosorbent assay (ELISA),western blot, immunohistochemical analysis.

Radio-immunoassay (RIA): In one version, this method involvesprecipitation of the desired substrate, e.g., LOR-1, with a specificantibody and radiolabelled antibody binding protein (e.g., protein Alabeled with I¹²⁵) immobilized on a precipitable carrier such as agarosebeads. The number of counts in the precipitated pellet is proportionalto the amount of substrate.

In an alternate version of the RIA, a labeled substrate and anunlabelled antibody binding protein are employed. A sample containing anunknown amount of substrate is added in varying amounts. The decrease inprecipitated counts from the labeled substrate is proportional to theamount of substrate in the added sample.

Enzyme linked immunosorbent assay (ELISA): This method involves fixationof a sample (e.g., fixed cells or a proteinaceous solution) containing aprotein substrate (e.g., LOR-1) to a surface such as a well of amicrotiter plate. A substrate specific antibody coupled to an enzyme isapplied and allowed to bind to the substrate. Presence of the antibodyis then detected and quantitated by a colorimetric reaction employingthe enzyme coupled to the antibody. Enzymes commonly employed in thismethod include horseradish peroxidase and alkaline phosphatase. If wellcalibrated and within the linear range of response, the amount ofsubstrate present in the sample is proportional to the amount of colorproduced. A substrate standard is generally employed to improvequantitative accuracy.

Western blot analysis: This method involves separation of a substrate(e.g., LOR-1 protein) from other proteins by means of an acrylamide gelfollowed by transfer of the substrate to a membrane (e.g., nylon orPVDF). Presence of the substrate is then detected by antibodies specificto the substrate, which are in turn detected by antibody bindingreagents. Antibody binding reagents may be, for example, protein A, orother antibodies. Antibody binding reagents may be radiolabelled orenzyme linked as described hereinabove. Detection may be byautoradiography, colorimetric reaction or chemiluminescence. This methodallows both quantitation of an amount of substrate and determination ofits identity by a relative position on the membrane which is indicativeof a migration distance in the acrylamide gel during electrophoresis.

Immunohistochemical analysis: This method involves detection of asubstrate in situ in fixed tissue by substrate specific antibodies. Thesubstrate specific antibodies may be enzyme linked or linked tofluorophores. Detection is by microscopy and subjective evaluation. Ifenzyme linked antibodies are employed, a colorimetric reaction may berequired.

Since tissue levels of a polypeptide can be inferred from the levels ofmRNA encoding such a polypeptide, the method according to this aspect ofthe present invention can also employ various polynucleotide detectionapproaches for determining the tissue level of the polypeptide of thepresent invention.

RNA molecules can be detected using methods known in the art includingfor example, Northern blot analysis, RT-PCR analyses, RNA in situhybridization stain and in situ RT-PCR stain.

Northern Blot analysis: This method involves the detection of aparticular RNA (e.g., the RNA molecule encoding LOR-1) in a mixture ofRNAs. An RNA sample is denatured by treatment with an agent (e.g.,formaldehyde) that prevents hydrogen bonding between base pairs,ensuring that all the RNA molecules have an unfolded, linearconformation. The individual RNA molecules are then separated accordingto size by gel electrophoresis and transferred to a nitrocellulose or anylon-based membrane to which the denatured RNAs adhere. The membrane isthen exposed to labeled DNA probes. Probes may be labeled usingradio-isotopes or enzyme linked nucleotides. Detection may be usingautoradiography, colorimetric reaction or chemiluminescence as describedhereinabove. This method allows both quantitation of an amount ofparticular RNA molecules and determination of its identity by a relativeposition on the membrane which is indicative of a migration distance inthe gel during electrophoresis.

RT-PCR analysis: This method uses PCR amplification of relatively rareRNAs molecules. First, RNA molecules from a particular tissue (e.g., acolon tumor tissue) are purified and converted into complementary DNA(cDNA) using a reverse transcriptase enzyme (such as an MMLV-RT) andprimers such as, oligo dT, random hexamers or gene specific primers, allof which are available from Invitrogen Life Technologies, Frederick,Md., USA. Then by applying gene specific primers and Taq DNA polymerase,a PCR amplification reaction is carried out in a PCR machine. Those ofskills in the art are capable of selecting the length and sequence ofthe gene specific primers and the PCR conditions (i.e., annealingtemperatures, number of cycles and the like) which are suitable fordetecting specific RNA molecules.

RNA in situ hybridization stain: In this method DNA or RNA probes areattached to the RNA molecules present in the tissue. Generally, a tissuesample (e.g., a colon tissue) is fixed to preserve its structure and toprevent the RNA from being degraded and then sectioned for microscopyand placed on a slide. Alternatively, frozen tissue samples can be firstsectioned and put on a slide and then subject to fixation prior tohybridization. Hybridization conditions include reagents such asformamide and salts (e.g., sodium chloride and sodium citrate) whichenable specific hybridization of the DNA or RNA probes with their targetmRNA molecules in situ while avoiding non-specific binding of probe.Those of skills in the art are capable of adjusting the hybridizationconditions (i.e., temperature, concentration of salts and formamide andthe like) to specific probes and types of cells. Followinghybridization, any unbound probe is washed off and the slide issubjected to either a photographic emulsion which reveals signalsgenerated using radio-labeled probes or to a colorimetric reaction whichreveals signals generated using enzyme-linked labeled probes asdescribed hereinabove.

In situ RT-PCR stain: This method is described in Nuovo G J, et al.(Intracellular localization of polymerase chain reaction (PCR)-amplifiedhepatitis C cDNA. Am J Surg Pathol. 1993, 17: 683-90) and Komminoth P,et al. [Evaluation of methods for hepatitis C virus detection inarchival liver biopsies. Comparison of histology, immunohistochemistry,in situ hybridization, reverse transcriptase polymerase chain reaction(RT-PCR) and in situ RT-PCR. Pathol Res Pract. 1994, 190: 1017-25].Briefly, the RT-PCR reaction is performed on fixed tissue sections byincorporating labeled nucleotides to the PCR reaction. The reaction iscarried on using a specific in situ RT-PCR apparatus such as thelaser-capture microdissection PixCell I LCM system available fromArcturus Engineering (Mountainview, Calif.).

Determination of an activity level of the polypeptide of the presentinvention (e.g., LOR-1) in a colon tumor tissue may be effected usingsuitable substrates in a cytochemical stain and/or in vitro activityassays.

Cytochemical stain: According to this method, a chromogenic substrate isapplied on the colon tumor tissue containing an active enzyme (e.g.,LOR-1). The enzyme catalyzes a reaction in which the substrate isdecomposed to produce a chromogenic product visible by a light or afluorescent microscope.

In vitro activity assays: In these methods the activity of a particularenzyme is measured in a protein mixture extracted from the tissue ofinterest (e.g., a colon tumor tissue). The activity can be measured in aspectrophotometer well using colorimetric methods (see for example,Wande Li, et al. Localization and activity of lysyl oxidase withinnuclei of fibrogenic cells. Proc. Natl. Acad. Sci. USA. 1997, 94:12817-12822) or can be measured in a non-denaturing acrylamide gel(i.e., activity gel). Following electrophoresis the gel is soaked in asolution containing a substrate and colorimetric reagents. The resultingstained band corresponds to the enzymatic activity of the polypeptide ofinterest (e.g., LOR-1). If well calibrated and within the linear rangeof response, the amount of enzyme present in the sample is proportionalto the amount of color produced. An enzyme standard is generallyemployed to improve quantitative accuracy.

Once the tissue level and/or the activity level of the polypeptide (ormRNA) of the present invention (e.g., LOR-1) is determined in the colontumor tissue the malignancy of the tumor is assessed by comparing theexpression level and/or activity in the colon tumor tissue to that of anormal colon tissue.

It will be appreciated that the normal colon tissue may be obtained froma biopsy and/or a surgery of a colon tissue obtained form a healthyindividual. Alternatively, the normal colon tissue can be obtained froman unaffected segment of the colon of the same individual. Methods ofdetermining the status of a normal colon tissue are known to skilled inthe art and include for example, a morphological evaluation of tissuesections.

Once malignancy of colon cancer is determined as described above, tissuelevel and/or activity level of the polypeptide (or mRNA thereof) of thepresent invention can also be utilized to stage the colon tumor and tothereby predict the prognosis of an individual diagnosed with coloncancer.

Such staging can be effected by assessing the tissue level and/oractivity level of the polypeptide and correlating it to results obtainedfrom colon cancer tissue at various stages (obtainable throughpathological evaluation of colon tumors). It will be appreciated thatsuch accurate and rapid staging will enable accurate and rapid prognosisof an individual afflicted with colon cancer and timely administrationof suitable treatment regimen.

Additional objects, advantages, and novel features of the presentinvention will become apparent to one ordinarily skilled in the art uponexamination of the following examples, which are not intended to belimiting. Additionally, each of the various embodiments and aspects ofthe present invention as delineated hereinabove and as claimed in theclaims section below finds experimental support in the followingexamples.

EXAMPLES

Reference is now made to the following examples, which together with theabove descriptions, illustrate the invention in a non limiting fashion.

Generally, the nomenclature used herein and the laboratory proceduresutilized in the present invention include molecular, biochemical,microbiological and recombinant DNA techniques. Such techniques arethoroughly explained in the literature. See, for example, “MolecularCloning: A laboratory Manual” Sambrook et al., (1989); “CurrentProtocols in Molecular Biology” Volumes I-III Ausubel, R. M., ed.(1994); Ausubel et al., “Current Protocols in Molecular Biology”, JohnWiley and Sons, Baltimore, Md. (1989); Perbal, “A Practical Guide toMolecular Cloning”, John Wiley & Sons, New York (1988); Watson et al.,“Recombinant DNA”, Scientific American Books, New York; Birren et al.(Eds.) “Genome Analysis: A Laboratory Manual Series”, Vols. 1-4, ColdSpring Harbor Laboratory Press, New York (1998); methodologies as setforth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and5,272,057; “Cell Biology: A Laboratory Handbook”, Volumes I-III Cellis,J. E., ed. (1994); “Current Protocols in Immunology” Volumes I-IIIColigan J. E., ed. (1994); Stites et al. (Eds), “Basic and ClinicalImmunology” (8th Edition), Appleton & Lange, Norwalk, Conn. (1994);Mishell and Shiigi (Eds), “Selected Methods in Cellular Immunology”, W.H. Freeman and Co., New York (1980); available immunoassays areextensively described in the patent and scientific literature, see, forexample, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578;3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533;3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521;“Oligonucleotide Synthesis” Gait, M. J., ed. (1984); “Nucleic AcidHybridization” Hames, B. D., and Higgins S. J., eds. (1985);“Transcription and Translation” Hames, B. D., and Higgins S. J., eds.(1984); “Animal Cell Culture” Freshney, R. I., ed. (1986); “ImmobilizedCells and Enzymes” IRL Press, (1986); “A Practical Guide to MolecularCloning” Perbal, B., (1984) and “Methods in Enzymology” Vol. 1-317,Academic Press; “PCR Protocols: A Guide To Methods And Applications”,Academic Press, San Diego, Calif. (1990); Marshak et al., “Strategiesfor Protein Purification and Characterization—A Laboratory CourseManual” CSHL Press (1996); all of which are incorporated by reference asif fully set forth herein. Other general references are providedthroughout this document. The procedures therein are believed to be wellknown in the art and are provided for the convenience of the reader. Allthe information contained therein is incorporated herein by reference.

Example 1 The Role of LOR1 in Angiogenesis

A study was conducted in efforts to further substantiate andcharacterize the role of LOR-1 in angiogenesis.

Materials and Methods

Human recombinant platelet factor-4 (PF4, GenBank Accession numberM20901) which was produced in bacteria and subsequently refolded wassupplied by Dr. Maione of Repligen Corp. (Boston, USA). Estrogen slowrelease pellets were obtained from Innovative Research of America,Sarasota, Fla., USA.

Construction of LOR-1 expression vector, transfection into MCF-7 cells,and expression: The LOR-1 cDNA (SEQ ID NO:1) was cloned into apCDNA3.1-hygro expression vector (Invitrogen Inc., USA) under thecontrol of a CMV promoter. Clones of cells expressing LOR-1 wereselected using hygromycine and assayed for LOR-1 expression using thepolyclonal antisera described below.

Construction of platelet factor-4 affinity columns and purification ofLOR-1 on such columns: PF4 was coupled to sepharose using a modificationof the method of Miron and Wilchek as previously described for vascularendothelial growth factor. Serum free conditioned medium was collectedfrom ³⁵S-methionine labeled MCF-7 cells which over-expressed LOR-1. Theconditioned medium was passed through the column twice. The column waswashed with phosphate buffered saline (300 mM NaCl, pH-7.2) and elutedwith PBS (containing 2M NaCl).

Experiments with nude mice: Modified or parental MCF-7 cells (10⁷ cellsper animal) were implanted under the skin of nude mice. A pellet of slowrelease estrogen was implanted 1 cm away as previously described. Tumorswere measured periodically, following which, tumors at least 1 cm insize were removed and immuno-histologically analyzed using a commercialantibody directed against a factor 8 like antigen which served as aspecific marker for endothelial cells.

In-situ hybridization: Fragments encompassing nucleotides 922-1564 ofLOR-1, nucleotides 976-1391 of LOL, nucleotides 400-950 of LO andnucleotides 1061-1590 of LOR-2 (as numbered from the ATG codon of thesesequences) where each independently subcloned into the Bluescript SK andKS (Strategene) vectors. A DIG cRNA labeling kit of Boehringer-Mannheimwas used to transcribe sense (s) and antisense (as) digoxigenin-labeledcRNA probes from the T7 promoter of the Bluescript constructs.Hybridization and subsequent detection of hybridized probes was carriedout essentially as previously described (Cohen et al., 2001).

Anti-LOR-1 polyclonal antisera: Antisera was generated by injecting arecombinant peptide containing the C-terminal 200 amino-acids of LOR-1(amino acids 540-744 of SEQ ID NO:2) into female rabbits. Serum wascollected 10 days following each injection and an immunoglobulinfraction was purified using a protein A Sepharose affinity column(Pharmacia).

Results

LOR-1 purification: A PF4 affinity column was used to detect endothelialcell proteins which specifically interact with PF4.

Two PF4 binding proteins were detected in conditioned medium of humanumbilical vein endothelial cells (HUVEC), whereas PF4 binding proteinswere not detected in detergent extracts of endothelial cells.

Two liters of conditioned medium enabled a partial purification of onesuch binding protein which eluted from the column at a relatively highsalt concentrations (0.4-0.5 M NaCl).

Further purification of this protein was performed using reverse phasehigh pressure liquid chromatography and SDS/PAGE chromatography. The PF4binding protein did not bind to heparin nor was it a heparan-sulfateproteoglycan since heparinase digestion failed to change its mobility inSDS/PAGE experiments.

Partial sequencing and database comparison revealed that the PF4 bindingprotein of the present invention (LOR-1) belongs to a family of proteinscontaining a lysyl oxidase like domain (Kim et al., 1995; Kim et al.,1999). Lysyl oxidases are copper dependent enzymes that participate inthe synthesis of the extracellular matrix by catalyzing the formation ofcovalent bonds between lysines of adjacent collagen or elastin fibers.

The full length amino acid sequence of LOR-1 (as deduced from theisolated cDNA sequence) displayed a high degree of identity to WS9-14, aprotein over expressed in senescent fibroblasts, in several types ofadherent cells (but not in non-adherent cells) and in fibroblasts, inwhich it was correlated to pro-collagen I-al expression levels, as wellas being induced by TGF-β and inhibited by phorbol esters and retinoicacid (Saito et al., 1997).

The role of LOR-1 in tumor development: Recombinant LOR-1 expressed inPAE cells specifically bound with the PF4 affinity column (FIG. 1).Since LOR-1 is a member of the LO family it was hypothesized that itparticipates in ECM formation during angiogenesis. Furthermore it wasalso hypothesized that PF4 suppresses or inhibits the pro-angiogenicactivity of LOR-1 thus inhibiting the later stages of blood vesselformation and as a result limiting tumor growth.

LOR-1 expression: In-situ hybridization demonstrated that LOR-1 isexpressed in a wide variety of tissues and cell types includingfibroblasts, adipocytes, nerve cells, endothelial cells and a variety ofepithelial cells. Several cell types, such as liver hepatocytes, did notexpress LOR-1; of the 4 LO family members examined (all except for LoxC)LOR-1 was the only one expressed in endothelial cells of blood vessels.

LOR-1 and cancer: as shown in FIG. 2, a direct correlation between theexpression levels of LOR-1 and the metastatic properties of breastcancer derived cell lines was demonstrated herein.

Since the epithelial cells which line the milk ducts of normal breasttissue (from which most breast tumors arise) express large amounts ofLOR-1 it is possible that the less metastatic lines lost LOR-1expression rather than gained it.

To substantiate its role in metastasis, LOR-1 cDNA was expressed innon-metastatic breast cancer derived MCF-7 cell lines which do notnormally express LOR-1. The expression of LOR-1 was examined usingrabbit polyclonal antibodies generated as described above (FIG. 3).

A control cell line which was transfected with an empty expressionvector, and an MCF-7 cell line expressing LOR-1 were implanted under theskin of immune deficient mice along with an estrogen slow release pelletas described above. Estrogen was added since the development of tumorsfrom this non-metastatic cell line is estrogen dependent.

The rate of tumor development in the mice was continuously monitored(FIG. 4); tumors 1 cm in size were excised and subjected to histologicalanalysis as described above. Interestingly, the rate of tumordevelopment varied between the two cell lines, with some tumorsexhibiting slower growth in the LOR-1 expressing MCF-7 and yet otherexhibiting slower growth in the control cells.

In order to overcome expression level problems, LOR-1 cDNA was placedunder the control of a tetracycline induced promoter (The TET-offsystem). Such a construct will enable to determine conclusively whetherthe reduced rate of tumor growth observed in LOR-1 expressing cells isindeed caused by LOR-1.

Tumors expressing large amounts of LOR-1 were sectioned and stained withan antibody directed against factor 8 like antigen, a specific marker ofendothelial cells. Control tumor tissue predominantly stained at thecapsule around the tumor while in the LOR-1 expressing tumors pronouncedstaining was observed in inner regions of the tumor tissue (FIG. 5a andFIG. 5b respectively).

The role of LOR-1 in Wilson's disease and in other chronic liverdiseases: Normal and diseased liver tissue were probed with LOR-1 sense(FIGS. 6a and 6c ) and antisense probes (FIGS. 6b and 6d ). Normal livertissues expresses very low levels of LOR-1 (FIG. 6b ). However, fibroticliver tissues such as those observed in Wilson's disease, exhibit astrong increase in hepatocyte expression of LOR-1 (FIG. 6d ).

Expression of LOR-1 in chick embryos: FIG. 7 illustrates LOR-1expression of LOR-1 mRNA in blood vessels of a developing chick embryo.Whole-mount in-situ hybridization of 4 day old chick embryos revealedLOR-1 mRNA expression in blood vessels located in the amnion (arrow).

The Lysyl oxidase family: A homology comparison between five members ofthe lysyl oxidase family which includes the LO and LOL subfamily and theLOR-1 and LOR-2 subfamily revealed a strong homology at the C-terminalportion which includes the conserved lysyl oxidase motif. LOR-1 andLOR-2 are characterized by long N-terminal stretches which are not foundin LO and LOL.

Example 2 MCF-7 Breast Cancer Cells Expressing Recombinant Lysyl OxidaseRelated Protein-1 (LOR-1) Form Invasive Tumors Characterized byExtensive Fibrosis

A study was conducted in efforts to further substantiate andcharacterize the role of LOR-1 in inhibiting metastasis and tumorfibrosis.

Materials and Methods

Estrogen pellets (17β-estradiol, 0.72 mg/pellet, 60-days release) werefrom Innovative Research of America, Fla., USA. Masson Trichrome stainkit was purchased from Bio-Optica (Milano, Italy), ReverseTranscriptase, G418 were from GIBCO BRL (U.K.), Hygromycin B,Tetracycline hydrochloride, Reticulum stain kit fast green and Siriusred (direct red 80) were from Sigma (USA), Restriction enzymes, T4ligase were from New England Biolabs (USA). The bacterial expressionvector pQE-30 and the nickel affinity column were obtained from Qiagen(Germany), ³²p-dATP was purchased from NEN (USA). Monoclonal anticytokeratin-7 (CAM 5.2) coupled to FITC was acquired from BectonDickinson (USA), and monoclonal mouse anti vimentin (clone V9) waspurchased from DAKO Denmark). Anti FITC alkaline phosphates-conjugatedwas purchased from ROCHE (USA), CAS block, citrate and EDTA antigenretrieval buffers were from Zymed (USA).

Cell culture: MCF-7 breast cancer cells were kindly provided by Dr.Hadasa Degani (Weizmann Institute, Israel). The MDA-MB-435 breast cancercell line was kindly provided by Dr. Israel Vlodaysky (Technion,Israel). The MDA-MB-231 cells were kindly provided by Dr. MichaelKlagsbrun (Harvard University, USA). These cell lines were routinelycultured in Dulbecco's modified eagle medium supplemented withgentamicin, amphotericin, glutamine and 10% Fetal Calf Serum (FCS).Human umbilical vein derived endothelial cells were isolated andcultured as described. Tissue culture media, sera, and cell culturesupplements were from Beth-Haemek Biological Industries, Israel, or fromGibco-BRL. MCF-7 TetOff cells (Clontech, USA) containing thetetracycline trans-activator (tTA) were grown in DMEM medium containing10% Tet system approved fetal calf serum (Clontech), in the presence of100 μg/ml G418, 150 μg/ml Hygromycin B and 1 μg/ml Tetracyclin.

Cloning of the LOR1 and LOR2 cDNA: Total RNA (4 μg) from HUVEC cells(for LOR1) or melanoma cells (for LOR2) was reversed transcribed usingMMLV reverse transcriptase (GIBCO BRL) as described. The LOR-1 and LOR-2cDNAs were amplified using the Expand Long High Fidelity PCR system(ROCHE) and the following pairs of amplification primers: For LOR-1 SEQID NOs:10 and 11, and for LOR-2 SEQ ID NOs:12 and 13. The 2.3 kb cDNA ofLOR-1 (SEQ ID NO:1) and the 2.26KB of LOR-2 (SEQ ID NO:4) were subclonedinto the pGEM-T Easy vector (Promega) by T-A cloning.

Generation of polyclonal antibodies against human LOR1: A cDNA fragmentcontaining nucleotides 1641-2253 of LOR1 (SEQ ID NO:14) was amplifiedusing the Expand High Fidelity PCR kit and a pair of amplificationprimers (SEQ ID NOs:15 and 16). The PCR product was subcloned into thepGEM-T Easy vector (Promega) by T-A cloning.

A 613 bp LOR1 cDNA fragment (SEQ ID NO:14) was digested with the Sph-Iand Hind III restriction enzymes and ligated into the bacterialexpression vector pQE-30, which added an in-frame sequence encoding asix histidine (6× His) tag to the 5′ end of the insert. The resultingplasmid was used to produce a recombinant, 6× His tagged 23 kDa peptide(SEQ ID NO:17). The peptide was purified from bacterial cell extractsusing nickel affinity chromatography, and further purified usingSDS-PAGE. The gel was electroblotted onto nitrocellulose and the bandcontaining the peptide was cut out from the blot, solubilized in DMSO,and used to immunize rabbits. Antiserum was affinity purified onprotein-A sepharose followed by affinity purification on a column towhich the recombinant peptide was coupled using a previously describedmethod (Wilchek and Miron, 1982). The antibody was eluted from thecolumn using 0.1 M glycine at pH 3.

Transfections: To constitutively express LOR1, the full length LOR1 cDNA(SEQ ID NO:1), was digested out of the pGEM-T easy vector (Promega) withHind III and XbaI (which were incorporated into the primers used for thecloning of the LOR1 cDNA) and ligated into the mammalian expressionvector pcDNA3.1 Hygro (Invitrogen, USA) to generate the expressionvector pcDNA-LOR1. Empty pCDNA3.1 Hygro plasmid or pcDNA-LOR1 plasmid(10-20 μg) were stably transfected into MCF-7 cells usingelectroporation with a BioRad gene pulser (960 μF, 0.28 V). Stabletransfectants were selected using 300 μg/ml hygromycin B. Clonesexpressing recombinant LOR1 were obtained in two consecutive stabletransfections and screened for LOR1 expression using our anti-LOR-1polyclonal antibodies. Conditioned medium was collected after 48 hoursfrom transfected cells and LOR-1 expression was monitored using westernblot analysis (FIG. 10A).

To inducively express LOR1, full length LOR1 cDNA (SEQ ID NO:1) wascloned into the pTET-Splice vector (Clontech), which enables aninducible expression under the control of tetracycline (Tet off system).The pTET-Splice plasmid DNA was digested with Hind III and SpeI andligated to the 2.3 kb hLOR1 cDNA fragment which was rescued out of thepCDNA-LOR1 plasmid using Hind III and XbaI. The resultant plasmid, whichwas designated as pTET-LOR1, was co-transfected into MCF-7 TetOff cellsalong with pTK-Hygro at a ratio of 20:1 respectively. LOR-1 expressingcells were selected in medium containing 100 μg/ml G418, 150 μg/mlhygromycin B and 1 μg/ml tetracycline. Stable transfectants werescreened for inducible expression of LOR1 using western blot analysis 48hours following removal of the tetracycline from the growth media. Theclone having the highest induction levels in the absence of tetracyclineand lowest basal expression levels in the presence of tetracycline wasselected and designated MCF-7/Tet-LOR1.

C6 glioma cells were transfected and screened for LOR1 expression asdescribed above.

Northern blot Analysis: Total RNA was extracted from cultured cellsusing Tri-Reagent (MRC, Cincinnati) according to the manufacture'sinstructions. Total RNA (15 μg) was loaded on a 1.2% agarose gel andNorthern blot analysis was carried out as previously described. LOR-1and LOR-2 ³²P labeled cDNA fragments; nucleotides 1-660 of SEQ ID NO:18and 1061-1590 of SEQ ID NO:19 (respectively) were used as probes.

Protein blot analysis: Serum free conditioned media (40 μl) wasseparated on a 8% SDS-PAGE gel and the proteins were electroblotted ontoa nitrocellulose filter using semi-dry electroblotting. The filter wasblocked for 1 hour at room temperature with TBST buffer containing 10 mMTris-HCl (pH-7.0), 0.15 M NaCl, and 0.3% Tween-20 supplemented with 10%low fat milk. The filter was incubated over night at 4° C. with affinitypurified rabbit anti-LOR1 polyclonal antibody in TBST (1:2500). The blotwas subsequently washed 3 times in TBST and incubated with goatanti-rabbit IgG peroxidase-conjugated secondary antibodies for 1 hour atroom temperature. Bound antibody was visualized using the ECL detectionsystem (Biological Industries, Israel).

Nude Mice Experiments: Slow release pellets containing 17β-estradiol(0.72 mg/pellet, 60 day release, Innovative Research) were pre-implantedsubcutaneously in 6-8 weeks old female athymic nude mice (CD1). MCF-7cells (10⁷ cells/mouse) were injected into the mammary fat pads. Tumorsize was measured with a caliper once or twice a week, and tumor volumewas determined using the following formula: volume=width²×length×0.52.Mice were sacrificed 4 weeks following injection of the MCF-7 cells. Inother experiments, the tumors were excised when reaching a diameter of0.8 cm. The primary tumor, the liver and the lungs were removed,weighted, fixed in 10% buffered formalin and embedded in paraffin.

The development of tumors from C6 glioma cells expressing recombinantLOR-1 was also studied. Cells (2×10⁵ cells/animal) transfected with acontrol expression vector or with an expression vector containing LOR1cDNA were injected subcutaneously in the hind limb. Mice were sacrificed3 weeks after the injection of the cells. The primary tumors wereremoved, fixed in 10% buffered formalin, and embedded in paraffin foranalysis.

Histology and Immunohistochemistry: Formalin-fixed, paraffin-embeddedtissues were cut into serial sections of 5μm each and used forimmunohistochemistry. Sections were deparaffinized by heating to 60° C.for 1 hour, washed twice with xylen for 5 min. and rehydrated byconsecutive washes in 100%, 95%, and 70% ethanol followed by a wash inwater. Endogenous peroxidase activity was inhibited by a 15 minuteincubation with 3% hydrogen peroxide in methanol, followed by washeswith water and PBS. The sections were then antigen retrieved by heatingtwice for 10 minutes in a microwave oven to 90° C. in citrate buffer atpH-6.2 (for cytokeratin and vimentin antibodies) or in 1mM EDTA buffer(for LOR1 antibody); blocking was performed using CAS block (Zymed).Following blocking, the sections were incubated for 1.5 hr at roomtemperature with the following antibodies, all diluted with in antibodydiluent reagent solution (Zymed): affinity purified anti-LOR1 antibody(1:30-1:50), monoclonal anti human cytokeratin-7 antibodies-FITCconjugated (1:50), or with monoclonal antibodies directed againstvimentin (1:50). The sections were then washed 3 times with TBST, andsecondary detection was applied using anti FITC alkalinephosphates-conjugate (Roche) at a 1:200 dilution (for anti cytokeratin)or DAKO Envision detection system (anti rabbit or anti mouse—HRP).Sections were developed in 3-amino-9-ethylcarbazole (AEC, DAKO)solution, counterstained by Hematoxylin, and photographed under amicroscope. In control experiments the primary antibodies were omitted.Masson Trichrome and reticulum stains were according to manufacturer'sprotocol. For Sirius red stain sections were incubated for 5 min. with0.03% (w/v) fast green solution, rinsed twice with 1% acetic acid,incubated for 15 min. in 0.1% Sirius red solution, rinsed as above,dehydrated and examined under polarizing light microscope.

Experimental Results

LOR-1 is expressed in highly metastatic breast cancer derived cell typesbut not in non-metastatic MCF-7 cells: Desmoplasia and formation offibrotic foci in breast cancer tumors is associated with the transitionfrom a localized, relatively benign tumor to an invasive/metastatictumor. Lysyl oxidases contribute to the deposition of collagen bycovalently cross-linking collagen monomers. To find out whetherexpression of lysyl oxidases is associated with the invasive/metastaticphenotype several human breast cancer derived cell types have beenscreened for the expression of lysyl oxidases. Northern blot analysisrevealed that the LOR-1 gene is expressed in the highly malignant,hormone independent MDA-MB-231 and MDA-MB-435 cells but not in hormonedependent non-metastatic MCF-7 cells (FIG. 9a ). LOR-2 on the other handwas expressed only in MDA-MB-435 cells but not in the MDA-MB-231 or inMCF-7 cells (FIG. 9b ).

Highly malignant grade 1 breast carcinoma cells do not express LOR-1while highly malignant cells of grade 3 carcinomas express LOR-1: LOR-1expression was confined to the milk ducts in both normal breast (FIG. 9c) and in in-situ ductal carcinoma (FIG. 9d ). However, in grade 1well-differentiated ductal breast carcinoma, wherein the tumor cellsmigrate out of the ducts to form pseudo ducts, the cells do not expressLOR-1 (FIG. 9e , black arrows). On the other hand, the cells of grade 3ductal breast carcinoma tumors, a highly malignant tumor characterizedby desmoplasia, express high levels of LOR-1 (FIG. 9f ).

Tumors generated in mice from LOR-1 MCF-7 transfected cells have aslower growth rate: In order to determine whether LOR-1 expressioncontributes to the progression of breast tumors and to theinvasive/metastatic phenotype, non-invasive MCF-7 cells have beentransfected with an expression plasmid containing LOR-1 cDNA (SEQ IDNO:1). Several transfectant clones have been isolated and selected fortheir LOR-1 expression. The conditioned medium of two LOR-1 expressingclones (clone 12 and 24) and of a clone transfected with an expressionvector alone (vec) was assayed with LOR-1 antibodies directed againstthe C-terminal portion of LOR-1 (SEQ ID NO:17). Western blot analysisrevealed higher levels of LOR-1 expression in clone 12 cells as comparedwith clone 24 cells, and no expression in cells transfected with theexpression vector alone (FIG. 10a ).

In addition, both LOR-1 expressing cells displayed extra protein bandsof about 70 kDa (FIGS. 10a, b ) suggesting that LOR-1 may undergoproteolytic processing like other members of the lysyl oxidase family.To verify that the low molecular weight forms are produced as a resultof post-translational processing, LOR-1 cDNA has been expressed in MCF-7cells under the control of a Tetracycline inducible promoter. It can beseen that once the tetracycline inhibition is removed, the cells startto produce full length LOR-1 which is then converted into a shorter, 70kDa C-terminal containing form (FIG. 10b ). It is not yet clear whetherall of these forms are enzymatically active.

To substantiate its role in tumor growth and metastasis LOR-1 producingcells and cells transfected with expression vector alone werepre-implanted subcutaneously in nude mice (described above).Interestingly, the growth rate of tumors containing LOR-1 expressingcells was retarded as compared with that of tumors developed fromparental cells or cells transfected with the expression vector alone(FIGS. 10c, d ).

To determine if the decreased tumor growth rate was a result of slowerproliferation, the proliferation rate of empty vector transfected MCF-7with that of clone 12 and clone 24 cells were also compared; significantdifferences in their rates of proliferation was not detected.

Tumors that develop from LOR-1 producing MCF-7 cells contain manynecrotic and fibrotic foci rich in collagen deposits: Hematoxylin-eosinstaining of tumor sections revealed major necrosis in tumors generatedfrom LOR-1 expressing MCF-7 cells (FIG. 11b ) and only a few necroticareas in tumors generated from parental or MCF-7 cells transfected withexpression vector alone (FIG. 11a ).

LOR-1 expressing tumors also contained extensive fibrotic areas mainlycomposed of host derived cells such as mouse fibroblasts rather thanMCF-7 cells. These cells are easily distinguishable from the host cellssince they do not react with an antibody against human keratin 7 (FIG.11c ).

Since LOR-1 was shown to oxidize lysine residues on collagen-I it wasanticipated that it may induce collagen cross-linking and deposition.However, this activity is probably depended on the existence of collagensince LOR-1 expressing MCF-7 cells grown in culture do not produce morecollagen than parental MCF-7 cells. To further understand theinvolvement of LOR-1 in tumor progression tumors derived from LOR-1expressing MCF-7 cells have been stained with Mason's Trichrome, areagent that reacts primarily with collagen-I and produces an azurecolor (Pinder et al., 1994). (1) Tumors that developed from parental orMCF-7 cells transfected by an expression vector alone contained limitedamounts of collagen that was scattered between the tumor cells (FIG. 11d, arrows). In-contrast, tumors that developed from clone 12 or clone 24LOR-1 expressing MCF-7 cells contained denser deposits of collagenbetween the tumor cells (FIG. 11e ). Furthermore, the fibrotic andnecrotic areas in these tumors appeared choke full with collagen fibers(FIG. 11f ). In addition, while the blood vessels within tumors thatdeveloped from vector-transfected MCF-7 cells remained almost unstained(FIG. 11g , arrows), the blood vessels in tumors derived from LOR-1expressing MCF-7 cells were sheathed by a thick layer of collagen (FIG.11h , arrows).

These experiments indicate that LOR-1 affects both collagen-I productionand deposition in MCF-7 derived breast cancer tumors. To substantiatethe involvement of LOR-1 in collagen deposition C6 glioma cells werealso subcutaneously injected into mice. While tumors generated fromC6-glioma cells did not contain large amounts of collagen, tumorsgenerated from LOR-1 expressing C6-glioma cells were rich with collagen.These results indicate a more general effect for LOR-1 on collagen-Ideposits in tumors. In addition, Reticulum staining of collagen-IIIrevealed that tumors generated from LOR-1 expressing MCF-7 or C6-gliomacells contained thicker and higher concentrations of collagen-III fibers(FIG. 12b, 12d ) as compared with tumors generated from cellstransfected with the expression vector alone (FIGS. 12a, c ).

Expression of LOR-1 in MCF-7 cells transforms the cells into invasivecells in-vivo: It was reported that the appearance of fibrotic foci inbreast cancer tumors correlates with their degree of invasiveness. Tosubstantiate the involvement of LOR-1 in tumor invasiveness humankeratin-7 staining was employed. Tumors generated from MCF-7 cellstransfected by the expression vector alone were surrounded by thickcapsules with sharp borders. No staining of human keratin-7 was observedwithin the capsule (FIG. 13a ) or in between blood vessels, nerves andmuscles located adjacent to the capsules (FIG. 13b , arrows). Incontrast, in tumors generated from LOR-1 expressing MCF-7 cells, humankeratin-7 positive cells were observed within the capsule (FIG. 13c ).Furthermore, in many areas the tumor cells migrated on-mass through thecapsule and invaded muscles (FIG. 13d ), nerves (FIG. 13e ) and bloodvessels (FIG. 13f ). The invading cells were identified as thetransfected LOR-1 expressing MCF-7 cells using an anti-LOR-1 antibody(FIG. 13g ). These observations provide a strong evidence that theproduction of LOR-1 by breast cancer tumor cells contribute to thetransition from localized non-invasive tumors to invasive tumors.Furthermore, aggregates of tumor cells were also detected inside lymphvessels adjacent to the tumors indicating that the LOR-1 expressingMCF-7 cells are metastatic (Luna, 1968).

Example 3 LOR-1 Expression is Correlated with the Malignancy of ColonTumors

To study the correlation between LOR-1 expression and the malignancy ofcolon tumors various colon tumor sections were subjected toimmunohistochemistry staining using an antibody directed against LOR-1.

Experimental Results

LOR-1 is moderately expressed in benign colon tumors: Normal colontissues and benign colon tumors including hyperplasia and adenomatissues were subjected to immunohistochemistry using an antibodydirected against the C-terminal of human LOR-1. As is shown in FIGS. 14a-b, while low level of LOR-1 expression was seen in a few cells of thenormal colon tissue (FIG. 14a ), a moderate level of expression wasdetected in the hyperplasia tissue and a substantial level of expressionwas detected in cells comprising the adenoma tissue of the benign colontumors (FIG. 14b ). Thus, these results demonstrate that the expressionof LOR-1 correlates with the formation of benign colon tumors.

Highly malignant colon tumors express high levels of LOR-1: To furthersubstantiate the correlation between LOR-1 and the progression of colontumors, low- and high-grade colon adenocarcinoma tissues were subjectedto LOR-1 immunohistochemistry. As is shown in FIGS. 14c -d, a high levelof LOR-1 expression was detected in both low-grade and high-gradeadenocarcinoma tissues. However, while in low-grade adenocarcinoma theexpression of LOR-1 was mainly confined to the carcinoma structures,(FIG. 14c , arrows), in the more malignant, high-grade colonadenocarcinoma high levels of LOR-1 expression was detected throughoutthe disorganized tumor tissue. These results demonstrate that a highlevel of LOR-1 expression is correlated with more malignant colontumors.

Altogether these results demonstrate that LOR-1 expression is correlatedwith the progression of colon cancer and suggest the use of LOR-1 indetermining the staging of colon cancer tumors.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable subcombination.

Although the invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives, modificationsand variations will be apparent to those skilled in the art.Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims. All publications, patents, patent applicationsand sequences identified by their accession numbers mentioned in thisspecification are herein incorporated in their entirety by referenceinto the specification, to the same extent as if each individualpublication, patent, patent application or sequence identified by theiraccession number was specifically and individually indicated to beincorporated herein by reference. In addition, citation oridentification of any reference in this application shall not beconstrued as an admission that such reference is available as prior artto the present invention.

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1. A method of determining that a colon tumor is metastatic, comprising:(a) obtaining a first sample of colon tumor tissue; (b) obtaining asecond sample of colon tumor tissue, which is non-metastatic; and (c)determining that a tissue level and/or an activity level of apolypeptide having an amino acid sequence set forth in SEQ ID NO: 2 ishigher in the first colon tumor tissue sample than in the second colontumor tissue sample; thereby determining that the tumor from which thefirst sample was derived is metastatic.
 2. The method of claim 1,wherein said determining in step (c) is effected by performing a methodselected from the group consisting of an immunological detection method,an RNA detection method, and an enzymatic activity detection method. 3.The method of claim 2, wherein said determining in step (c) is effectedby performing the immunological detection method, which is selected fromthe group consisting of a radio-immunoassay (RIA), an enzyme linkedimmunosorbent assay (ELISA), a Western blot analysis, and animmunohistochemical analysis.
 4. The method of claim 2, wherein saiddetermining in step (c) is effected by performing the RNA detectionmethod, which is selected from the group consisting of a Northern blotanalysis, an RNA in situ hybridization stain, an RT-PCR analysis, and anin situ RT-PCR stain.
 5. The method of claim 2, wherein said determiningin step (c) is effected by performing the enzymatic activity detectionmethod, which is selected from the group consisting of a cytochemicalstain, an in vitro activity assay, and an activity gel.
 6. The method ofclaim 1, wherein said determining in step (c) comprises contacting thesamples with an antibody that specifically binds the polypeptide,whereby a polypeptide in one or both of the samples forms a complex withthe antibody, and detecting the antibody-polypeptide complex orcomplexes so formed.
 7. The method of claim 1, wherein said determiningin step (c) comprises contacting the samples with a nucleic acid probehomologous to a human LOR-1 gene, whereby an LOR-1 mRNA in one or bothof the samples forms a hybrid with the probe, and detecting the hybridor hybrids so formed.
 8. A method of determining whether a colon tumoris metastatic, comprising: determining a tissue level and/or an activitylevel of a polypeptide having an amino acid sequence set forth in SEQ IDNO: 2 in a test colon tumor tissue sample; wherein a higher tissue leveland/or activity level of said polypeptide in the test colon tumor tissuesample, compared with that of a non-metastatic colon tumor tissuesample, indicates that the tumor from which the test colon tumor tissuesample was derived is metastatic.
 9. The method of claim 8, wherein saiddetermining is effected by performing a method selected from the groupconsisting of an immunological detection method, an RNA detectionmethod, and an enzymatic activity detection method.
 10. The method ofclaim 9, wherein: said immunological detection method is selected fromthe group consisting of a radio-immunoassay (RIA), an enzyme linkedimmunosorbent assay (ELISA), a Western blot analysis, and animmunohistochemical analysis; said RNA detection method is selected fromthe group consisting of a Northern blot analysis, an RNA in situhybridization stain, an RT-PCR analysis, and an in situ RT-PCR stain; orsaid enzymatic activity detection method is selected from the groupconsisting of a cytochemical stain, an in vitro activity assay, and anactivity gel.
 11. The method of claim 8, wherein said determiningcomprises contacting the test colon tumor sample with an antibody thatspecifically binds the polypeptide, whereby a polypeptide in the sampleforms a complex with the antibody, and detecting theantibody-polypeptide complex so formed.
 12. The method of claim 8,wherein said determining comprises contacting the test colon tumorsample with a nucleic acid probe homologous to a human LOR-1 gene,whereby an LOR-1 mRNA in the sample forms a hybrid with the probe, anddetecting the hybrid so formed.
 13. The method of claim 8, furthercomprising first obtaining the test colon tumor sample from a subject.14. A method of determining that a colon tumor is metastatic,comprising: determining that a tissue level and/or activity level of apolypeptide having an amino acid sequence set forth in SEQ ID NO: 2 in asample from the colon tumor is higher than a control tissue and/oractivity level of the polypeptide, thereby determining that the colontumor is metastatic.
 15. The method of claim 14, wherein the controltissue and/or activity level is a tissue and/or activity level of thepolypeptide in a non-metastatic colon tumor sample.
 16. The method ofclaim 14, wherein said determining is effected by assaying the samplefrom the colon tumor.
 17. The method of claim 16, wherein said assayingis effected by a method selected from the group consisting of animmunological detection method, an RNA detection method, and anenzymatic activity detection method.
 18. The method of claim 17,wherein: said immunological detection method is selected from the groupconsisting of a radio-immunoassay (RIA), an enzyme linked immunosorbentassay (ELISA), a Western blot analysis, and an immunohistochemicalanalysis; said RNA detection method is selected from the groupconsisting of a Northern blot analysis, an RNA in situ hybridizationstain, an RT-PCR analysis, and an in situ RT-PCR stain; or saidenzymatic activity detection method is selected from the groupconsisting of a cytochemical stain, an in vitro activity assay, and anactivity gel.
 19. The method of claim 16, wherein said assayingcomprises contacting the sample from the colon tumor with an antibodythat specifically binds the polypeptide, whereby a polypeptide in thesample forms a complex with the antibody, and detecting theantibody-polypeptide complex so formed.
 20. The method of claim 16,wherein said assaying comprises contacting the sample from the colontumor with a nucleic acid probe homologous to a human LOR-1 gene,whereby an LOR-1 mRNA in the sample forms a hybrid with the probe, anddetecting the hybrid so formed.